JP2003516150A - Full-length human cDNA encoding a cryptic secretory protein - Google Patents

Abstract

  (57) [Summary] The present invention relates to GENSET polynucleotides and polypeptides. Such GENSET products can be used as reagents in forensic analysis, as chromosomal markers in expression vector production, tissue / cell / organelle specific markers. In addition, they can be used in screening and diagnostic assays for screening compounds that can be used to treat abnormal GENSET expression and / or biological activity, and GENSET-related disorders.

Description

Detailed Description of the Invention

Related Applications This application claims the priority of US Provisional Application Nos. 60 / 169,629 and 60 / 187,470, filed December 8, 1999 and March 6, 2000, respectively, the entire disclosure of which is hereby incorporated by reference. It is incorporated as a reference in the book.

FIELD OF THE INVENTION The present invention relates to GENSET polypeptides, fragments thereof, and 5 of the GENSET genes.
It relates to polynucleotides encoding regulatory regions located at the'and 3'ends.
The invention also relates to the polypeptides encoded by the GENSET polynucleotides and fragments thereof. The invention also provides a recombinant vector comprising a polynucleotide of the invention, in particular a recombinant vector comprising a sequence encoding a GENSET control region or a GENSET polypeptide, and a host cell comprising the polynucleotide of the invention, and such a vector. And a method of host cell production. The invention further relates to the use of the recombinant vectors and host cells described above in the production of the polypeptides of the invention. The invention further relates to antibodies that specifically bind the polypeptides of the invention, and methods of producing such antibodies and fragments thereof. The invention also provides methods of detecting the presence of the polynucleotides and polypeptides of the invention in a sample, methods of diagnosing and screening for abnormal GENSET gene expression and / or biological activity, and modulating GENSET gene activity or expression. Methods of screening compounds for potency as well as uses of such compounds are provided. BACKGROUND OF THE INVENTION The putative 50,000-100,000 genes scattered on the human chromosome offer the potential for understanding human disease and its diagnosis and treatment. In addition, a probe that can specifically hybridize with loci dispersed throughout the human genome has a use in constructing a high-resolution chromosome map and identifying an individual. Currently, two approaches are pursued for the purpose of identifying and characterizing genes distributed along the human genome. In one method, large fragments of genomic DNA are isolated, cloned and sequenced. Potential open reading frames contained in these genomic sequences are identified using bioinformatics software. However, in order to discover protein-coding sequences distributed throughout the genome with this method, human DNA that does not
Must be sequenced for a large fragment of. Extensive sequencing is required,
Bioinformatics software can also make erroneous characterizations of the resulting genomic sequences (ie, labeling non-coding DNA as coding DNA and vice versa).

[0002] Another method uses more direct means to identify and characterize human genes. In this method, complementary DNA (cDNA) is synthesized from isolated messenger RNA (mRNA) that encodes a human protein. If you use this method,
Sequencing may be performed only on the DNA derived from the protein coding fragment of the genome. Traditionally, the above cDNAs, often short EST sequences, have been obtained from oligo dT primed cDNA libraries. Therefore, they corresponded mainly to the 3'untranslated region of the mRNA. For one thing, since the general technique for obtaining cDNA is not suitable for isolating the cDNA sequence derived from the 5'end of mRNA, ES derived from the 3'end of mRNA is
Mainly T-sequence (Adams et al., Nature 377: 3-174, 1996, Hillier et al., Genome Re
s. 6: 807-828, 1996). In addition, in the reported case that a long cDNA sequence was obtained,
The reported sequence generally corresponds to the coding sequence and is the complete sequence of the mRNA derived from the cDNA.
Does not include the 5'untranslated region (5'UTR). In particular, the 5'UTR has been shown to affect mRNA stability or translation. Thus, for example, regulation of gene expression may be achieved via a selective 5'UTR, as shown for translation of tissue inhibitors of metalloprotease mRNA contained in mitogenic activated cells (Waterhouse et al., J Biol. Chem
.265: 5585-9. 1990). Furthermore, modification of the 5'UTR by events such as mutations, insertions or translocations may even manifest itself in the development of the disease. For example, Fragile X Syndrome, the most common cause of inherited mental retardation, has multiple CGG trinucleotides inserted into the 5'UTR of fragile X mRNA, resulting in
Partly due to the inhibition of protein synthesis due to ribosome stalling (Feng et al., Science 268: 731-4, 1995). Aberrant mutations in the 5'UTR region known to inhibit translation of the proto-oncogene c-myc may result in upregulation of c-myc protein levels in cells from patients with multiple myeloma Shown (Willis et al., Curr Top Microbiol Immunol 224: 269-7.
6, 1997). Also, using a cDNA library primed with oligo dT,
The complete 5'UTR cannot be isolated because the incomplete sequence obtained in this way may not contain the first exon of the mRNA, especially if the first exon is short. In addition, some exons located upstream of the splice site (often short exons) may not be included. Therefore, there is a demand for obtaining a sequence derived from the 5'end of mRNA. In addition, it was obtained in a large sequencing project (Adams et al., N.
ature 377: 174, 1996, Hillier et al., Genome Res 6: 807-828, 1996) Despite the large amount of EST data, there is limited information on the biological function of the mRNA corresponding to the cDNA obtained from it. It is clear that there is.

[0003] In particular, ESTs yield only partial coding sequences, although knowledge of the complete coding sequence is absolutely necessary for investigating the biological function of mRNA. To date, there have been only limited successes in large-scale full-length cDNA cloning because the methods for constructing full-length cDNA libraries are very inefficient. In particular, such methods require large amounts of mRNA (Ederly et al.
1995) either makes the full-length library less representative when only a small amount of tissue is available, or requires PCR amplification to obtain a significant number of clones (Mar.
uyama et al., 1994; CLONTECHniques, 1996), resulting in a highly biased cDNA library with long and rare cDNAs lost. Therefore, the total length
There is a need to obtain a cDNA, that is, a cDNA containing the complete coding sequence for its corresponding mRNA. This application presents a number of cDNAs, called GENSET polynucleotides, isolated from a full-length cDNA library, obtained by the method described in WO 00/37491. Although many sequences from the human chromosome have practical applications, methods based on the identification and characterization of chromosomal sequences encoding protein products are particularly relevant for diagnostic and therapeutic applications. Of the 50,000 to 100,000 protein-encoding genes, the genes encoding proteins secreted by cells in which the proteins are synthesized as well as the secreted proteins themselves are particularly useful as potential therapeutic agents. Such proteins are often involved in cell-cell communication and may be responsible for clinically relevant responses in their target cells. In fact, tissue plasminogen activator,
Several secreted proteins are currently in clinical use, including G-CSF, GM-CSF, erythropoietin, human growth hormone, insulin, interferon-α, interferon-β, interferon-γ and interleukin-2. These proteins are used to treat a wide range of conditions, including acute myocardial infarction, acute ischemic stroke, anemia, diabetes, growth hormone deficiency, hepatitis, kidney cancer, chemotherapy-induced neutropenia and multiple sclerosis. It is used. For these reasons, cDNAs encoding secreted proteins or fragments thereof represent a particularly important source of potential therapeutic agents. Therefore, there is a need to identify and characterize secreted proteins and the nucleic acids that encode them.

[0004] In addition to their therapeutic utility in their own right, secretory proteins contain short peptides called signal peptides that direct secretion at the amino terminus. These signal peptides are encoded by a signal sequence located at the 5'end of the coding sequence of the gene encoding the secreted protein. These signal peptides direct extracellular secretion of proteins to which they are operably linked, so by operably linking a signal sequence to the gene encoding the protein for which secretion is desired,
Signal sequences can be utilized to direct the efficient secretion of proteins. Also, fragments of the signal peptide, called transmembrane sequences, may be used to direct intracellular transfer of the peptide or protein of interest. This method may be successful in gene therapy strategies where it is desirable to deliver a particular gene product to cells other than those producing the gene product. A signal sequence encoding a signal peptide can also be applied to simplify the protein purification method.
In such applications, extracellular secretion of the desired protein greatly reduces purification, reducing the number of unwanted proteins that must be distinguished from the desired protein. Therefore, for secretory proteins encoding signal peptides, there is a need to identify and characterize the 5'fragments of genes. The human protein-encoding sequence may also be used as a therapeutic or diagnostic agent. In particular, such sequences can be used to determine whether an individual is likely to develop a detectable phenotype, such as a disease, as a result of mutations in the protein coding sequence. If an individual is at risk of suffering from a disease or other undesirable phenotype as a result of mutations in such a coding sequence, gene therapy may be used to introduce the normal coding sequence into the desired coding sequence. Can correct non-phenotypes. Alternatively, where the unwanted phenotype is due to overexpression of the protein encoded by the coding sequence, antisense or triple helix based strategies can be used to reduce protein expression. The GENSET human polypeptide encoded by the coding sequence may also be utilized as a therapeutic by direct administration to an individual who is in a condition such as a disease caused by a mutation in the sequence encoding the polypeptide. In such cases, the condition may be cured or alleviated by administering the polypeptide to the individual.

Human polypeptides or fragments thereof can also be used to produce antibodies useful in determining the tissue type or species of origin of biological samples. Antibodies may also be used to determine the subcellular localization of human secretory polypeptides or the localization of polypeptides fused to human polypeptides. The antibody may also be used in an immunoaffinity chromatography method to isolate, purify or concentrate the human polypeptide or a target polypeptide fused to a human polypeptide. Very limited information is available regarding the number of human genes for which the promoter and upstream regulatory regions have already been identified and characterized. This may be partly due to the difficulty in isolating such regulatory sequences. Upstream control sequences, such as transcription factor binding sites, are generally too short to serve as probes to isolate promoters from human genomic libraries. Recently, several methods have been developed for isolating human promoters. One of them is CpG
It consists of creating an island library (Cross et al., Nature Genetics 6: 236-244, 1994). The second method consists of utilizing a SpeI binding protein to isolate a human genomic DNA sequence containing the SpeI binding site (Mortlock et al., Genome Res.
6: 327-335, 1996). Both methods have limitations due to lack of specificity and comprehensiveness. Therefore, there is a need to identify as well as systematically characterize 5'fragments of genes. To efficiently identify and isolate a cDNA containing the 5'end of the corresponding mRNA, the 5'UTR and upstream regulatory regions that control the location, developmental stage, rate and amount of protein synthesis, and mRNA stability. Can be used for Once identified and characterized, these regulatory regions can be utilized in gene therapy or protein purification programs to synthesize proteins in desired amounts and positions, or to inhibit, reduce or prevent unwanted gene product synthesis. The cDNA containing the 5'end of the protein gene may contain a sequence useful as a probe for chromosome mapping and individual identification. Therefore, there is a need to identify and characterize sequences upstream of the 5'coding sequences of protein-encoding genes.

SUMMARY OF THE INVENTION The present invention provides a composition comprising a purified or isolated polynucleotide comprising, consisting of, or consisting essentially of, a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 1-241 sequences; (b) sequence of the clone insert of the deposited clone pool; (c) complete coding sequence of SEQ ID NOs: 1-241; (d) complete coding sequence of the clone insert of the deposited clone pool; (e) SEQ ID NO: A sequence encoding one of the polypeptides 242 to 482; (f) a sequence encoding one of the polypeptides encoded by the clone inserts of the deposited clone pool; (g) GENS.
Genomic sequence encoding ET polypeptide; (h) 5'transcription control region of GENSET gene; (i) 3'transcription control region of GENSET gene; (j) (g) to (i) any nucleotide A polynucleotide comprising a sequence; (k) (a)-(j
(1) the polynucleotide is a single chain, a double chain, or a part is a single chain and a part is a double chain;
To (k) a polynucleotide comprising a nucleotide sequence; (m) a polynucleotide comprising a nucleotide sequence that is complementary to any of the single-stranded polynucleotides (l). The present invention further provides fragments of the nucleic acid molecules of (a)-(m) above. The invention also provides biologically active forms, variants, fragments and derivatives of the protein, where "biologically active" means that the form, variant, fragment or derivative is known in the art or herein. It is shown to have detectable activity in any of the described in vitro assays, or to have detectable function in vivo. In a preferred embodiment, the determination of whether a particular polypeptide is biologically active is based on any of the specific assays or functional characteristics set forth below for each protein of the invention. . Accordingly, one embodiment of the present invention is selected from the group consisting of the sequences of SEQ ID NOs: 1-241 and the sequences of clone inserts of the deposited clone pool, their complementary sequences, allelic variants thereof, and degenerate variants thereof. A composition comprising a purified or isolated nucleic acid, which comprises the sequence In one aspect of this embodiment, the nucleic acid is recombinant.
Another embodiment of the invention is a sequence selected from the group consisting of the sequences SEQ ID NOs: 1-241 and the sequence of the clone insert of the deposited clone pool, its complementary sequence, its allelic variants, and its degenerate variants. And a purified or isolated nucleic acid comprising at least 8 contiguous nucleotides. In one aspect of this embodiment, the nucleic acid is at least 10, 12, 15, 18, 20, 25, 28, which is the selected sequence, its complementary sequence, its allelic variants, and its degenerate variants. 30, 35, 40, 50
, 75, 100, 150, 200, 300, 400, 500, 800, 1000, 1500 or 2000 consecutive nucleotides. The nucleic acid may be a recombinant nucleic acid.

Another embodiment of the present invention is from the group consisting of the polynucleotide of the present invention under stringent conditions, preferably the sequences of SEQ ID NOs: 1-241 and the sequence of the clone insert of the deposited clone pool, its complementary sequence. A sequence of at least 15, 18, 20, 23, 25, 28, 30, 35, 40, which hybridizes with any of the selected sequences.
A composition comprising a vertebrate purified or isolated nucleic acid which is 50, 75, 100, 200, 300, 500, 1000 or 2000 nucleotides. In one aspect of this embodiment, the nucleic acid is recombinant. Another embodiment of the invention is a purified or single sequence comprising the complete coding sequence of a sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-241 and the clone insert of the deposited clone pool, or allelic variants thereof. A composition comprising isolated nucleic acid. In one aspect of this embodiment, the nucleic acid is recombinant. Yet another embodiment of the present invention is selected from the group consisting of the sequences SEQ ID NOs: 1-31 and 33-143 and the sequence of a clone insert encoding a secreted protein in the deposited clone pool, or an allelic variant thereof. A composition comprising purified or isolated nucleic acid comprising a contiguous span of sequences, wherein said contiguous span encodes a mature protein. In one aspect of this embodiment, the nucleic acid is recombinant. In another aspect of this embodiment, the nucleic acid is an expression vector in which the flanking spans encoding the mature protein are operably linked to a promoter. Yet another embodiment of the present invention is selected from the group consisting of the sequences SEQ ID NOs: 1-31 and 33-143 and the sequence of a clone insert encoding a secreted protein in the deposited clone pool, or an allelic variant thereof. A composition comprising purified or isolated nucleic acid comprising a contiguous span of sequences, wherein said contiguous span encodes a signal peptide. In one aspect of this embodiment, the nucleic acid is recombinant. In another aspect of this embodiment, the nucleic acid is a fusion vector in which said flanking span encoding a signal peptide is operably linked to a second nucleic acid encoding a heterologous polypeptide. Another embodiment of the invention is a purified or encoded polypeptide comprising a sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-241 and the clone insert of the deposited clone pool, or allelic variants thereof. A composition comprising an isolated nucleic acid. In one aspect of this embodiment, the nucleic acid is recombinant.

Another embodiment of the invention is from the group consisting of the sequences SEQ ID NOs: 1-31 and 33-143 and the sequence of a clone insert encoding a secreted protein in the deposited clone pool, or an allelic variant thereof. A composition comprising purified or isolated nucleic acid encoding a polypeptide comprising the sequence of a mature protein contained in the selected sequence. In one aspect of this embodiment, the nucleic acid is recombinant. Another embodiment of the invention is selected from the group consisting of the sequences of SEQ ID NOs: 1-31 and 33-143 and the sequence of a clone insert encoding a secreted protein in the deposited clone pool, or an allelic variant thereof. A composition comprising purified or isolated nucleic acid encoding a polypeptide comprising the sequence of a signal peptide contained in the sequence. In another aspect, it is present in the vector of the invention. A further embodiment of the present invention comprises a polynucleotide of the present invention and at least 70%.
, More preferably at least 75%, even more preferably at least 80%, 85%
, 90%, 95%, 96%, 97%, 98%, or 99% identical to a purified or isolated polynucleotide. Methods of determining identity include those widely known in the art and described herein. Such analysis can be performed using full length polynucleotide sequences or sequences of any length. For example, for any two sequences, whether one is a protein or both are proteins, 10, 25, 50, 100, 250, 500, 1000
, 2000 or more flanking nucleotide regions can be compared. Also, any two sequences are, for example, at least about 10, 25, 50, 100, 250, 500.
, 1000, or more regions of contiguous nucleotides can be identified as homologous even if they share sequence homology with the restricted region of either polynucleotide. The present invention comprises an amino acid sequence selected from the group consisting of:
Alternatively, further provided is a composition comprising, predominantly, a purified or isolated polypeptide: (a) a polypeptide of SEQ ID NOS: 242-482; (b) a polypeptide encoded by a clonal insert of the deposited clone pool; (c). SEQ ID NOs: 242-482
(D) an epitope-bearing fragment of the polypeptide encoded by the clone insert contained in the deposited clone pool; (e) a polypeptide domain of SEQ ID NO: 242-482;
f) a domain of the polypeptide encoded by the clone insert contained in the deposited clone pool; (g) an allelic variant polypeptide of any of the polypeptides of (a)-(f). The invention further provides fragments of the polypeptides of (a)-(g) above that are biologically active or comprise a biological functional domain.

Another embodiment of the invention comprises a sequence selected from the group consisting of the sequences SEQ ID NOs: 242-482 and the polypeptide sequences encoded by the clone inserts of the deposited clone pool, or allelic variants thereof. , A composition containing a purified or isolated protein. Another embodiment of this invention is a sequence selected from the group consisting of the sequences SEQ ID NOs: 242-482 and the clone insert of the deposited clone pool, or an allelic variant thereof, at least 5 , A composition comprising a purified or isolated polypeptide comprising 6 or 8 contiguous amino acids. In one aspect of this embodiment, the purified or isolated polypeptide is at least 10, 12, 15, 20, 25, 30, 35, 40, which is a selected sequence or allelic variant thereof.
, 50, 60, 75, 100, 150, 200, 250, 300, 350, 400, 450 or 500 consecutive amino acids. Another embodiment of the invention is a sequence selected from the group consisting of the sequences SEQ ID NOs: 242-272 and 274-384, and the polypeptide sequence encoded by the clonal insert of the deposited clone pool, or allelic variants thereof. A composition comprising a purified or isolated polypeptide containing the signal peptide of. Yet another embodiment of the invention is selected from the group consisting of the sequences of SEQ ID NOs: 242-272 and 274-384, and the polypeptide sequence encoded by the clone insert of the deposited clone pool, or allelic variants thereof. A composition comprising a purified or isolated polypeptide comprising a mature protein of sequence. A further embodiment of the present invention comprises a polynucleotide of the present invention and at least 70%.
, And more preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%
, Or a polypeptide having an amino acid sequence with 99% similarity, and a polynucleotide of the invention, at least 70%, more preferably at least 75%,
Even more preferably at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,
Or a composition comprising a polypeptide having an amino acid sequence that is 99% identical.
Such analyzes can be performed using full length polypeptide sequences or sequences of any length. For example, for any two sequences, whether one is a protein or both are proteins, 10, 25, 50, 100, 250, 500, 1000
, 2000 or more flanking amino acid regions can be compared. Also, any two sequences may be, for example, at least about 10, 25, 50, 100, 250, 500, 100.
Regions of zero or more contiguous amino acids can be identified as homologous even if they share sequence homology with the restricted regions of either protein. Compositions containing purified or isolated nucleic acid molecules encoding such polypeptides are also included in the invention. Methods of determining identity include those widely known in the art and described herein.

The invention also relates to compositions comprising a recombinant vector comprising a purified or isolated polynucleotide of the invention, and host cells that are recombinant for the polynucleotide of the invention, as well as such vectors and host cells. Regarding the method of creation. The invention further relates to the use of the above recombinant vector and recombinant host cells in the production of GENSET polypeptides. As a result, another embodiment of the present invention is a vector containing any of the polynucleotides of the present invention. In a preferred embodiment, the vector is a nucleic acid sequence encoding a polypeptide selected from the group consisting of the sequences SEQ ID NOs: 242-482 and the clone insert of the deposited clone pool, or allelic variants thereof. And an expression vector comprising the nucleic acid sequence operably linked to a promoter. In another preferred embodiment, the vector is
Encodes a signal peptide selected from the group consisting of the sequences of SEQ ID NOs: 242-272 and 274-384, and the secretory polypeptide sequence encoded by the clone insert of the deposited clone pool, or an allelic variant thereof. A secretory vector comprising a nucleic acid sequence, wherein said nucleic acid sequence is operably linked to a heterologous protein and thus said signal peptide directs secretion of said heterologous protein. Yet another embodiment of the invention is a method of making a protein comprising a sequence selected from the group consisting of the sequences SEQ ID NOs: 242-482, and the polypeptide sequence encoded by the clone insert of the deposited clone pool, The method comprises the steps of: a) obtaining a cDNA comprising a sequence selected from the group consisting of the sequences SEQ ID NOS: 1-241 and the sequences of the clone inserts of the deposited clone pool; b) said cDNA being operative with a promoter Inserting the cDNA into an expression vector so that it is ligated; and c) introducing the expression vector into a host cell, whereby the host cell
Producing a protein encoded by NA. In one aspect of this embodiment, the method further comprises the step of isolating said protein.

Another embodiment of the invention is a protein obtainable by the method described in the paragraph above. Another embodiment of the invention is the amino acid of the mature protein contained in a sequence selected from the group consisting of the sequences SEQ ID NOs: 242-272 and 274-384, and the polypeptide sequence encoded by the clone insert of the deposited clone pool. A method of producing a protein comprising a sequence, said method comprising the steps of: a) a sequence selected from the group consisting of the sequences SEQ ID NOs: 1-31 and 33-143 and the sequence of the clone insert of the deposited clone pool. A cDNA encoding a mature protein; b) inserting the cDNA into an expression vector such that the cDNA is operably linked to a promoter; and c) expressing the cDNA. The vector is introduced into a host cell whereby the host cell
Producing a mature protein encoded by NA. In one aspect of this embodiment, the method further comprises the step of isolating said protein. Another embodiment of the invention is a mature protein obtainable by the method described in the paragraph above. Other embodiments of the present invention include a sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-241, and the clone inserts of the deposited clone pool, or their complementary sequences described herein, which may be purified or A composition comprising a host cell containing an isolated nucleic acid. Another embodiment of the invention is a host cell containing purified or isolated nucleic acid, which comprises the complete coding sequence of a sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-241 and the sequences of the clone inserts of the deposited clone pool. It is a composition containing. Another embodiment of the invention is a purified or isolated nucleic acid comprising flanking spans consisting of sequences selected from the group consisting of the sequences of SEQ ID NOs: 1-31 and 33-143, and the sequences of clone inserts of the deposited clone pool. A composition comprising a host cell comprising Another embodiment of the invention is a purified or isolated nucleic acid comprising flanking spans consisting of sequences selected from the group consisting of the sequences of SEQ ID NOs: 1-31 and 33-143, and the sequences of clone inserts of the deposited clone pool. A composition comprising a host cell comprising: wherein said flanking span encodes a signal peptide.

The invention further relates to other methods of making the polypeptides of the invention. The invention further relates to a transgenic plant or animal, wherein said transgenic plant or animal is transgenic for a polynucleotide of the invention and expresses a polypeptide of the invention. The invention further relates to compositions comprising antibodies and fragments thereof that specifically bind to the GENSET polypeptides of the invention, and methods of producing such antibodies and fragments thereof. Therefore, another embodiment of the present invention is to specifically bind to a protein comprising a sequence selected from the group consisting of the sequences SEQ ID NOs: 242-482 and the polypeptide sequence encoded by the clone insert of the deposited clone pool. Is possible,
A composition containing a purified or isolated antibody. In one aspect of this embodiment, the antibody is
It is possible to bind to a polypeptide comprising at least 6 contiguous amino acids, at least 8 contiguous amino acids, or at least 10 contiguous amino acids which is the selected sequence. The invention also provides kits and methods for detecting GENSET gene expression and / or biological activity in a biological sample. One such method is GENSET
Assaying the expression of GENSET polynucleotides in biological samples using the polymerase chain reaction (PCR) to amplify and detect polynucleotides, or Southern and Northern for detecting GENSET genomic DNA, cDNA, or mRNA. Includes blot hybridization. Alternatively, the method of detecting GENSET gene expression in a test sample can be achieved by using a compound that binds to the GENSET polypeptide or a part of the GENSET polypeptide of the present invention. The present invention also contemplates therapeutic methods for suppressing or amplifying GENSET gene expression, respectively, diagnostic methods for identifying individuals or non-human animals exhibiting elevated or decreased GENSET product levels, and abnormal expression of the GENSET gene. Alternatively, it relates to a method of identifying an individual or non-human animal at high risk of developing, or currently having, certain diseases / disorders associated with biological activity. The invention also includes the ability to modulate (eg, increase or suppress) the activity or expression of the GENSET gene, including compounds that interact with the GENSET gene regulatory sequences and compounds that interact directly or indirectly with the GENSET polypeptide. about,
Kits and methods for screening compounds. The use of such compounds is also within the scope of the invention.

The present invention also relates to pharmacologically or physiologically tolerable compositions comprising the active substances, polypeptides, polynucleotides or antibodies of the invention. The invention also relates to computer systems comprising the cDNA and polypeptide codes of the sequences of the invention, and computer-related methods of comparing sequences and identifying homologies or characteristics using the GENSET sequences of the invention. In another aspect, the invention provides an isolated polynucleotide, which polynucleotide comprises i) one of the sequences set forth as SEQ ID NOS: 242-482 or a polypeptide sequence encoded by a clone insert of a deposited clone pool. A nucleic acid sequence comprising a polypeptide comprising an amino acid sequence having at least about 80% identity with one; or a biologically active fragment of said polypeptide. In one embodiment, the polypeptide comprises one of the sequences set forth as SEQ ID NOS: 242-482 or one of the polypeptide sequences encoded by the clone insert of the deposited clone pool. In other embodiments, the polypeptide comprises a signal peptide. In other embodiments, the polypeptide comprises a mature protein. In other embodiments, the nucleic acid sequence flanks at least about 100 of one of the sequences set forth as SEQ ID NOS: 1-241 or one of the sequences of the clone inserts of the deposited clone pool.
It has at least about 80% identity for nucleotides. In other embodiments,
The polynucleotide hybridizes under stringent conditions to a polynucleotide comprising one of the sequences set forth as SEQ ID NOS: 1-241 or one of the clone insert sequences of the deposited clone pool. In another embodiment, the nucleic acid sequence comprises one of the sequences set forth as SEQ ID NOS: 1-241 or one of the clone insert sequences of the deposited clone pool. In other embodiments, the polynucleotide is operably linked to a promoter. In another aspect, the invention provides an expression vector that comprises a polynucleotide operably linked to a promoter. In another aspect, the invention provides a host cell that is recombinant for the polynucleotide. In another aspect, the invention provides a non-human transgenic animal containing host cells. In another aspect, the invention provides a method of making a GENSET polypeptide, said method comprising a) providing a host cell population comprising a polynucleotide described herein, and b) said host. Culturing the host cell population under conditions which support the production of the polypeptide intracellularly.

In one embodiment, the method further comprises purifying the polypeptide from a host cell population. In another aspect, the invention provides a method of making a GENSET polypeptide, the method comprising: a) providing a population of cells comprising a polynucleotide described herein; b) the polypeptide in a cell. Culturing the cell population under conditions that support the production of C .; and c) purifying the polypeptide from the cell population. In another aspect, the invention provides an isolated polynucleotide, wherein said polynucleotide flanks at least about 100 of one of the sequences set forth as SEQ ID NOS: 1-241 or one of the clone insert sequences of the deposited clone pool. It comprises nucleic acid sequences having at least about 80% identity with respect to nucleotides. In one embodiment, the polynucleotide hybridizes under stringent conditions to a polynucleotide comprising one of the sequences set forth as SEQ ID NOS: 1-241 or one of the clone insert sequences of the deposited clone pool. In another embodiment, the polynucleotide comprises one of the sequences set forth as SEQ ID NOS: 1-241 or one of the clone insert sequences of the deposited clone pool. In another aspect, the invention provides a bioactive polypeptide encoded by any of the polynucleotides described herein. In another aspect, the invention provides an isolated polypeptide or biologically active fragment thereof, wherein said polypeptide is a polypeptide encoded by one of the sequences set forth as SEQ ID NOS: 242-482 or a clonal insert of a deposited clone pool. It comprises an amino acid sequence having at least about 80% sequence identity with one of the peptide sequences. In one embodiment, the polypeptide is selectively recognized by an antibody raised against an antigenic polypeptide, or an antigenic fragment thereof, said antigenic polypeptide having the sequence set forth in SEQ ID NOs: 242-482. One or one of the polypeptide sequences encoded by the clone inserts of the deposited clone pool. In another embodiment, the polypeptide comprises one of the sequences set forth as SEQ ID NOS: 242-482 or one of the polypeptide sequences encoded by the clonal insert of the deposited clone pool. In other embodiments, the polypeptide comprises a signal peptide. In other embodiments, the polypeptide comprises a mature protein.

In another aspect, the invention provides an antibody that specifically binds to any of the polypeptides described herein. In another aspect, the invention provides a method of determining that the GENSET gene is expressed in a mammal, the method comprising the steps of: a) providing a biological sample from the mammal; b) contacting the biological sample with any of the following: i) a polynucleotide that hybridizes under stringent conditions to the polynucleotide of claim 1; or ii) the polypeptide of claim 19. A polypeptide that specifically binds; and c) a polynucleotide in the sample and
Detecting the presence or absence of hybridization between RNA species or the presence or absence of binding of a polypeptide to a protein in a sample; here, the detection of hybridization or binding means that the GENSET gene is expressed in a mammal. Indicates. In one embodiment, the polynucleotide is a primer and hybridization is detected by detecting the presence of an amplification product containing the primer sequence. In other embodiments, the polypeptide is an antibody. In another aspect, the invention provides a method of determining an increase or decrease in GENSET gene expression levels in a mammal, the method comprising the steps of: a) providing a biological sample from a mammal; and b). Comparing the amount of any of the polypeptides described herein, or RNA species encoding the polypeptide, in a biological sample to levels detected or expected in a control sample; An increase in the amount of polypeptide or RNA species in the biological sample compared to the expected level is indicative of elevated GENSET gene expression in mammals and was compared to the level detected or expected in the control sample. A decrease in the amount of polypeptide or RNA species in the biological sample indicates decreased GENSET gene expression in mammals. In another aspect, the invention provides a method of identifying a candidate modulator of a GENSET polypeptide, the method comprising: a) contacting a polypeptide described herein with a test compound. And b) determining that the compound specifically binds to the polypeptide; where detecting that the compound specifically binds to the polypeptide is that the compound is a candidate modulator of a GENSET polypeptide. Indicates that.

Brief Description of the Tables Table I shows the Applicant's internal designation numbers assigned to each SEQ ID NO, whether the sequence is a nucleic acid sequence or a polypeptide sequence, or the vector into which the cDNA was cloned. Table II shows the structural features of each cDNA of SEQ ID NOs: 1-241 (location of complete coding sequence, signal peptide, mature polypeptide, polyA signal and polyA site). Table III lists the variants of the cDNA of the present invention. Table IV shows the positions of fragments that are preferably excluded from the present invention. Tables Va and b show the fragment positions that are preferably excluded or included in the present invention. Tables IV and Va, and Table Vb show the inclusion and exclusion of polynucleotides independent of each other, in addition to those described in the specification, and thus are not meant to be limiting. Table VI lists the known biological structure and functional domains of the polypeptides of the invention. Table VII lists the antigenic peaks of predicted antigenic epitopes of the polypeptides of the invention. Table VIII lists the putative chromosomal locations of the polynucleotides of the invention. Table IX lists Genset cDNA libraries of tissues and cell types tested to express the polynucleotides of the invention. Table X relates to the bias in the spatial distribution of the polynucleotide sequences of the invention. Table XI lists the sub-predicted cellular levels of the cDNA of the invention. Table XII provides US priority applications, particularly polynucleotides according to US provisional patent application serial numbers 60 / 169,629 and 60/187 (columns entitled "Priority Application SEQ ID NOs") and polypeptides of the present application ("this application"). The correspondence between "SEQ ID NO:" and the column entitled) is shown. BRIEF DESCRIPTION OF SEQUENCE LISTINGS SEQ ID NOS: 1-31 and 33-143 are the nucleotide sequences of cDNAs encoding cryptic secretory proteins. The positions of the sequences encoding the ORF (open reading frame) and the signal peptide are listed in the accompanying sequence listing. Furthermore, the Heijne score of the signal peptide calculated as described below.
Is listed as a "score" in the accompanying sequence listing. Signal peptide sequences are listed as "sequences" in the accompanying sequence listing. "/" In the signal peptide sequence indicates a position where cleavage of the signal peptide by proteolysis occurs to give a mature protein. Where appropriate, the first and last nucleotide positions of the coding sequence are ultimately the first and last nucleotide positions of polyA, thus indicating the first and last nucleotide positions of the polyA site.

SEQ ID NOs: 32 and 144-241 are nucleotide sequences of cDNA for which the sequence encoding the signal peptide has not been identified to date. However, subsequent analysis may identify sequences encoding signal peptides in these nucleic acids. The location of the ORF is listed in the accompanying sequence listing. Where appropriate, the first and last nucleotide positions of the coding sequence are ultimately the first and last nucleotide positions of polyA, thus indicating the first and last nucleotide positions of the polyA site. SEQ ID NOs: 242-272 and 274-384 are the amino acid sequences of polypeptides including signal peptides. These polypeptides have SEQ ID NOs: 1-31 and 33-143.
Are encoded by the respective cDNAs. The positions of the signal peptide sequences are listed in the accompanying sequence listing. SEQ ID NOs: 273 and 385-482 are amino acid sequences of polypeptides for which no signal peptide has been identified to date. However, subsequent analysis may identify signal peptides in these polypeptides. These polypeptides are encoded by the nucleic acids of SEQ ID NOs: 32 and 144-241, respectively. In accordance with the regulations regarding sequence listing, the following codes are used in the sequence listing to describe nucleotide sequences. The code "r" in the sequence indicates that the nucleotide can be guanine or adenine. The code "y" in the array is
It indicates that the nucleotide can be thymine or cytosine. Code in array "
"m" indicates that the nucleotide can be adenine or cytosine. The code "k" in the sequence indicates that the nucleotide can be guanine or thymine. The code "s" in the sequence indicates that the nucleotide can be guanine or cytosine. The code "w" in the sequence indicates that the nucleotide can be adenine or thymine. Furthermore, in all cases where the symbol "n" is present in the nucleic acid sequence,
It means that the nucleotide can be adenine, guanine, cytosine, or thymine. In some cases, the polypeptide sequences in the sequence listing include the symbol "Xaa".
Applicants believe that these "Xaa" symbols should not be present (1) residues that cannot be identified due to nucleotide sequence ambiguity, or (2) (if more accurately sequenced) , A stop codon in a sequenced sequence. In some cases, the genetic code may suggest some possible identities for unknown amino acids.

In the case of secreted proteins, according to the management rules for sequence listing,
Encoding proteins (ie, proteins that include a signal peptide and a mature protein or a portion thereof) range from amino acid residues having a negative number to amino acid residues having a positive number. Therefore, the first amino acid of the mature protein by signal peptide cleavage is designated as amino acid number 1, and the first amino acid of the signal peptide is designated by an appropriate negative number. However, in this application, the position on the amino acid sequence is always shown on the full length polypeptide and the first amino acid of the signal peptide is designated as amino acid number 1. Definitions Before describing the present invention in detail, the following definitions are published in order to explain and define the meaning and scope of the terms used in the present specification to describe the present invention. The term "GENSET gene" as used herein includes genomic, mRNA and cDNA sequences encoding the GENSET protein, including the 5'and 3'untranslated regions of said sequence. As used herein, a "secreted" protein is a protein that is transported across or across a membrane when expressed in a suitable host cell and results from a signal peptide in its amino acid sequence. Including transportation. "Secreted" proteins include, but are not limited to, proteins that are completely (eg, soluble proteins) or partially (eg, receptors) secreted by the cells in which they are expressed. "Secreted" proteins also include, but are not limited to, proteins that are transported across the membrane of the endoplasmic reticulum. As used herein, a "mature protein" is a polypeptide fragment produced after cleavage of a signal peptide. "Full-length coding sequence" or open reading frame (OR of GENSET gene
F) as used herein means the complete coding sequence of said gene. In the case of a secreted protein, the full length coding sequence comprises the coding sequence for the signal peptide and the coding sequence for the mature polypeptide. Thus, by "full length polypeptide" is meant the complete polypeptide encoded by the GENSET gene, which, in the case of secreted proteins, includes both signal and mature polypeptides. The position of the full-length polypeptide and, in the case of secretory proteins, of the signal peptide and mature polypeptide are given in the sequence listing in the Appendix.

By “GENSET biological activity” is intended a polypeptide that exhibits an activity that is similar, but not necessarily identical to that of a GENSET polypeptide of the invention. The GENSET bioactivity of a given polypeptide can be assessed using the appropriate bioassay methods known to those of skill in the art as described herein. In contrast, "biological activity" means the activity that the polypeptide of the present invention may have. The "corresponding mRNA" is the mRNA that was the template for cDNA synthesis that produced the cDNA of the present invention.
Means By "corresponding genomic DNA" is meant genomic DNA encoding an mRNA containing the sequence of one of the strands of the cDNA, with thymidine residues in the sequence of the cDNA replaced by uracil residues of the mRNA. "Deposited clone pool", as used herein, refers to the pool of clones designated GENSET.07PRF deposited with the ATCC under accession number PTA-1218 on January 21, 2000. "Heterologous" as used herein is intended to designate a polynucleotide or polypeptide other than a GENSET polynucleotide or polypeptide, respectively. "Isolated" requires that the material be removed from its original environment (eg, the natural environment if it is of natural origin). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide isolated from some or all coexisting materials in the natural system is isolated. . Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, and the vector or composition may be It can be isolated because it is not part of its natural environment. For example, naturally occurring polynucleotides present in living animals are not isolated, but identical polynucleotides separated from some or all coexisting materials in the natural system are isolated. Excluded specifically from the definition of "isolated" are naturally occurring chromosomes (such as chromosomal nucleic acids), artificial chromosome libraries, genomic libraries, and in vitro nucleic acid preparations or transfected / transformed host cell preparations (wherein A host cell is a cDNA library present either as an in vitro xenogeneic preparation or seeded as a heterogeneous population that is a single colony). further,
The library is also specifically excluded if a particular polynucleotide is 5% or less of the nucleic acid inserts present in the vector molecule. Also excluded are whole cell genomic DNA or whole cell RNA preparations, including said whole cell preparations that have been subjected to mechanical shearing or enzymatic digestion. Furthermore, the polynucleotide of the present invention is not further separated from the heterologous polynucleotide in the electrophoretic medium (for example,
Either an in vitro preparation or a heterogeneous mixture separated by excision of a single band from a heterogeneous band population in an agarose gel or nylon blot), electrophoresis (including blot transfer of the like) In the case of, the whole cell preparation is specifically excluded.

“Purified” does not require absolute purity and is intended to be defined relatively. It is particularly expected to purify the starting material or natural material to at least one digit, preferably two or three digits, more preferably four or five digits. For example, when the concentration increases from 0.1% to 10%, it means that two-digit purification has been performed. For example, each cDNA clone isolated from a cDNA library was conventionally purified until electrophoretic homogeneity was obtained. The sequences obtained from these clones could not be obtained directly from either the library or total human DNA. cDNA clones do not occur naturally, but rather are obtained by the manipulation of partially purified naturally-occurring material (messenger RNA). The conversion of mRNA into a cDNA library involves the production of synthetic material (cDNA), and pure individual cDNA clones can be isolated from the synthetic library by clonal selection. Therefore, the generation of a cDNA library from messenger RNA, followed by isolation of individual clones from that library, results in about 10 ⁴ to 10 ⁶ fold purification of the native message. "Purified" further describes herein a polypeptide or polynucleotide of the invention that is separated from other compounds, including, but not limited to, polypeptides or polynucleotides, carbohydrates, lipids, etc. . "Purified" is sometimes used to identify separating a monomeric polypeptide of the invention from oligomeric forms such as homo, hetero, dimers, trimers. "
"Purified" may also be used to identify separating covalently closed polynucleotides from linear polynucleotides. A polynucleotide is substantially pure if at least 50%, preferably 60-75%, of the sample exhibits a single polynucleotide sequence and conformation (covalently closed to linear). Substantially pure polypeptide or polynucleotide is typically about 10% by weight of the polypeptide or polynucleotide sample, respectively.
50%, preferably 60-90%, more generally about 95% and preferably about 99% or more pure (where% is% by weight). The purity, or homogeneity, of polypeptides and polynucleotides is demonstrated by a number of means known in the art such as agarose or polyacrylamide gel electrophoresis of samples followed by single band observation by gel staining. For certain purposes, the use of HPLC or other means known in the art provides high resolution. In other embodiments, purification of polypeptides and polynucleotides of the invention may be expressed as "at least" percent purity with respect to heterologous polypeptides and polynucleotides (DNA, RNA, or both). In a preferred embodiment, the polypeptides and polynucleotides of the invention are at least: 10%, 20%, 30%, 40%, 50%, respectively for heterologous polypeptides and polynucleotides.
60%, 70%, 80%, 90%, 95%, 96%, 96%, 98%, 99%, or 100
% Purity. In a further preferred embodiment, the polypeptide and the polynucleotide are each in the range of any number between 90% and 100% and up to one thousandth with respect to either the heterologous polypeptide or polynucleotide (eg, at least 99.995). % Polypeptide or polynucleotide), or purity as a weight / weight ratio with respect to all compounds and molecules other than those present in the carrier. Each number up to 1/1000 that represents percent purity is considered an individual species of purity.

As used interchangeably herein, “nucleic acid molecule”, “oligonucleotide”, and “polynucleotide” refer to RNA or DNA (either single-stranded or double-stranded, coding , Complementary or antisense), or RNA / DNA hybrid sequences consisting of one or more nucleotides, either single-stranded or double-stranded, although each of the above species may be specifically identified. “Nucleotide” is used herein as an adjective to describe a molecule that includes single- or double-stranded RNA, DNA or RNA / DNA hybrid sequences regardless of length. More specifically, a "nucleotide sequence" includes the nuclear material itself, and thus the sequence information that characterizes the particular DNA or RNA molecule biochemically (ie, the order of letters selected among the four base letters). ) Is not limited to. "Nucleotide" is also used herein as a noun to refer to an individual nucleotide or variant of a nucleotide and means a molecule, or an individual unit contained in a large nucleic acid molecule, which is a purine or pyrimidine, ribose or deoxygen. It contains a ribose sugar moiety and a phosphate group or, in the case of a nucleotide in an oligonucleotide or polynucleotide, a phosphodiester bond. “Nucleotide” as used herein also refers to at least one such as (a) a sublinking group, (b) a purine analog, (c) a pyrimidine analog, or (d) a similar sugar. A “modified nucleotide” containing one modification is included. For illustrative examples of analogous linking groups, purines, pyrimidines, and sugars, see the examples in International Patent Application Publication WO 95/04064, which is incorporated herein by reference in its entirety. Preferred modifications of the invention are 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5
-Carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, (-D-galactosylcueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2 -Dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, (-D-mannosyl cuocosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v) ybutoxosine, pseudouracil, queocin, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3- (3- Amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine, but are not limited thereto. The polynucleotide sequences of the invention can be prepared utilizing any of the known methods, including synthetic, recombinant, ex vivo production, or combinations thereof, as well as any of the purification methods known in the art. it can. Methylenemethylimino linked oligonucleotides as well as mixed backbone compounds are described in US Pat. No. 5,378,82.
5; 5,386,023; 5,489,677; 5,602,240; and 5,610,289, the disclosures of which are incorporated herein by reference in their entireties. Formacetal and thioformacetal linked oligonucleotides can be prepared as described in US Pat. Nos. 5,264,562 and 5,264,564, the disclosures of which are incorporated herein by reference in their entireties.
Ethylene oxide linked oligonucleotides can be prepared as described in US Pat. No. 5,223,618, the disclosure of which is incorporated herein by reference in its entirety. Phosphinate oligonucleotides can be prepared as described in US Pat. No. 5,508,270, the disclosure of which is incorporated herein by reference in its entirety. Alkylphosphonate oligonucleotides can be prepared as described in US Pat. No. 4,469,863, the disclosure of which is incorporated herein by reference in its entirety. The 3'-deoxy-3'-methylenephosphonate oligonucleotides can be prepared as described in U.S. Pat.Nos. 5,610,289 and 5,625,050, the disclosures of which are incorporated herein by reference in their entirety. There is. Phosphoramidite oligonucleotides are described in US Pat.
It can be prepared as described in Nos. 775 and 5,366,878, the disclosure of which is incorporated herein by reference in its entirety. Alkylphosphonothionate oligonucleotides can be prepared as described in International Patent Application Publication Nos. WO 94/17093 and WO 94/02499, the entire texts of which are incorporated herein by reference. 3'-deoxy-3'-aminophosphoramidate oligonucleotides can be prepared as described in U.S. Pat.No. 5,476,925, the publication of which is incorporated herein by reference in its entirety. . Phosphate 3-ester oligonucleotides can be prepared as described in US Pat. No. 5,023,243, the disclosure of which is incorporated herein by reference in its entirety. Borano phosphate oligonucleotides are described in US Pat. No. 5,130,302 and
It can be prepared as described in 5,177,198, the disclosure of which is incorporated herein by reference in its entirety.

“Upstream”, as used herein, means a position in the 5 ′ end of a polynucleotide from a particular reference point. "Base-paired" and "Watson & Crick base-paired" are used interchangeably herein and are a thymine or uracil residue and three hydrogens attached to an adenine residue by two hydrogen bonds. It is meant that nucleotides that can hydrogen bond to each other are key to sequence identity in the manner found in double-stranded helix DNA with cytosine and guanine residues bound by a bond (Styer, 195.
5, the disclosure of which is incorporated by reference herein in its entirety). By “complementary” or “complement thereof” is meant herein a polynucleotide sequence in which the entire complementary region is in its entirety capable of forming Watson & Crick base pairs with another particular polynucleotide. For the purposes of the present invention, a first polynucleotide is considered to be complementary to a second polynucleotide if each base of the first polynucleotide is paired with its complementary base. Complementary bases are generally A and T (or A and
U), or C and G. As used herein, "complementary" is used as a synonym for "complementary polynucleotide,""complementary nucleic acid," and "complementary nucleotide sequence." These terms apply to a pair of polynucleotides based solely on their sequence, not to the particular set under which the two polynucleotides actually bind. Unless otherwise specified, all complementary polynucleotides are perfectly complementary over the entire length of the polynucleotide of interest. "Polypeptide" and "protein" are used interchangeably herein and refer to a polymer of amino acids regardless of polymer length. Thus, the definition of polypeptide includes peptides, oligopeptides and proteins. The term also identifies chemical or post-expression modifications of the polypeptides of the invention, although chemical or post-expression modifications of these polypeptides may be included or excluded in certain embodiments. Do not exclude. Thus, for example, modifications to a polypeptide that include covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide. Furthermore, polypeptides with these modifications may be identified as individual species and included or excluded from the invention. Natural or other chemical modifications, such as those listed in the examples above, can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, and may be cyclic with or without branching, for example, as a result of ubiquitin conjugation. Modifications are by acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavins, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives. Attachment, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-link formation, cysteine formation, pyroglutamate formation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, PE
Includes transfer-RNA-mediated addition of amino acids to proteins such as Gylation (polyethylene glycolation), proteolytic reactions, phosphorylation, prenylation, racemization, selenonization, sulphation, arginyl linkages and ubiquinin linkages. (For example, C
reighton (1993); Seifter et al. (1990); Rattan et al. (1992)) Furthermore, this definition also includes one or more amino acid analogs (eg, non-naturally occurring amino acids, unrelated biological systems). (Including amino acids that occur only in nature, modified amino acids of mammalian origin, etc.), polypeptides with substitution bonds, and other modifications of natural and non-natural origin known in the art. .

As used herein, "recombinant polynucleotide" and "polynucleotide construct" are used interchangeably and are artificially designed and at least not found as contiguous nucleotide sequences in their original natural environment. A linear or circular, purified or isolated polynucleotide comprising two nucleotide sequences. In particular, the term means that the polynucleotide or cDNA is flanked by "backbone" nucleic acids that are not flanked in their natural environment. In addition, "concentrated" means cD
It means that NA is 5% or more of the number of nucleic acid inserts in the population of nucleic acid backbone molecules. The backbone molecule according to the present invention comprises an expression vector, a self-replicating nucleic acid,
It includes nucleic acids such as viruses, integrated nucleic acids, and nucleic acids used to maintain or manipulate other vectors or nucleic acid inserts of interest. The enriched cDNA is preferably 15% or more of the number of nucleic acid inserts in the recombinant backbone molecule population. Concentrated c
More preferably, the DNA is 50% or more of the number of nucleic acid inserts in the recombinant backbone molecule population. In a particularly preferred embodiment, the enriched cDNA comprises 90% or more of the number of nucleic acid inserts in the recombinant backbone molecule population (any number between 90 and 100%, for example, 1 in 1000, such as 99.5%. Including up to the place of). By "recombinant polypeptide" as used herein is meant a polypeptide that is artificially designed and contains at least two polypeptide sequences that are not found as flanking polypeptide sequences in the original natural environment. Or a polypeptide expressed from a recombinant polynucleotide. As used herein, "operably linked" refers to the association of polynucleotide elements in a functional relationship. By a sequence "operably linked" to a control sequence, such as a promoter, is meant that the control elements are in the correct position and orientation with respect to the nucleic acid, which modulates initiation of RNA polymerase and expression of the nucleic acid of interest. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. As used herein, "non-human animal" means any non-human animal including insects, birds, rodents and other common mammals. Preferred non-human animals include: primates; pigs, goats, sheep, donkeys, cattle, horses, chickens, rabbits; and rodents, preferably rats or mice. As used herein, the term "animal" means
It means all species in the animal kingdom, preferably birds and fish, and more preferably vertebrates, including mammals. “Animal” and “mammal” actively embraces human subjects, unless otherwise specified as “non-human”.

By “domain” is meant an amino acid fragment with specific biological properties. The term encompasses all known structural and linear biological motifs. Examples of such motifs are leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitin binding sites, alpha helices, and beta sheets, signal peptides that direct protein secretion, post-translational modification sites, enzyme active sites. , Substrate binding sites and enzyme cleavage sites, but are not limited thereto. “Including”, “consisting of” and “consisting essentially of” have respective distinct meanings, but may be used interchangeably throughout the application. “Has” means “includes”
Has the same meaning as, and may be replaced with either “consisting of” or “consisting mainly of”. The “amplification product” means a product obtained by any amplification reaction such as PCR, RTPCR, LCR, and the like. A "modulator" of a protein or other compound is a physical binding to the protein, a change in the amount or quality of protein expression, a change in the activity, property or behavior of a measurable or detectable protein, or a protein or other By any agent that has a functional effect on a protein, including any interaction with a compound. A "test compound" can be a molecule that is evaluated for its ability to modulate a protein or other compound (activity modulating). Unless otherwise specified in this application, the polynucleotides and polypeptides of the invention, nucleotides and amino acids, respectively, are contiguous and free of heterologous sequence. Identity between nucleic acids or polypeptides "Percentage sequence identity" and "percentage homology" refer to
Used interchangeably herein, to mean a comparison between polynucleotides and polypeptides, measured by comparing two sequences that are optimally aligned on the comparison window, where A portion of a polynucleotide or polypeptide sequence may contain additions or deletions (ie gaps) when compared to a reference sequence (without additions or deletions) for optimal alignment between the two sequences. . The ratio is determined by measuring the number of positions that produce the same nucleobase or amino acid residue in both sequences and producing the number of match positions, and dividing the number of match positions by the total number of positions in the comparison window to obtain the sequence identity percentage. To obtain, it is calculated by multiplying the result by 100. Homology is evaluated using various sequence comparison algorithms and programs known in the art. Such algorithms and programs include TBLASTIN, BLASTP, FASTA,
Including but not limited to TFASTA, CLUSTALW, FASTDB (Pearson and
Lipman, 1988; Altschul et al., 1990; Thompson et al., 1994; Higgins et al., 1996; A
ltschul et al., 1990; Altschul et al., 1993; Brutlag et al., 1990). The full text of the publication is incorporated into the present specification as a reference.

In a particularly preferred embodiment, protein and nucleic acid sequence homology is determined by the basic local alignment search tool (“BLAST”) (Kar), which is known in the art.
lin and Altschul, 1990; Altschul et al., 1990, 1993, 1997), the disclosure of which is incorporated herein by reference in its entirety. In particular, 5
Two specific BLAST programs are used to perform the following tasks: (1) BLASTP and BLAST3 compare amino acid query sequences against protein sequence databases; (2) BLASTN compares nucleotide query sequences. (3) BLASTX compares the 6-frame conceptual translation product of the query nucleotide sequence (both strands) to the protein sequence database; (4) TBLASTN is the query protein sequence. Against all 6 open reading frames (both strands) translated nucleotide sequence database; and (5) TBLASTX compares the nucleotide query sequence 6 frame translation to the nucleotide sequence database 6 frame translation. Compare. The BLAST program is a "high score segment pair" between a query amino acid or nucleic acid sequence and a test sequence that is preferably obtained from a protein or nucleic acid sequence database.
The homologous sequences are identified by identifying similar segments, referred to herein as. High scoring segment pairs are preferably identified (ie, aligned) by means of a scoring matrix, many of which are known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., 1992; Henikoff and Henikoff, 1993), the publication of which is incorporated by reference in its entirety. It ’s not so good, but
PAM or PAM250 matrix may be used (eg Schwartz and Dayh
Off, eds., 1978), the entire disclosure of which is incorporated herein by reference. The BLAST program evaluates the statistical significance of all identified high-scoring segments and preferably selects those that meet a user-specified significance threshold, such as user-specified homology. Preferably, the statistical significance of the high score segment pair is determined by the Karlin's statistical significance formula (eg Karl
in and Altschul, 1990), the disclosure of which is incorporated herein by reference in its entirety. The BLAST program may be used with default parameters or modified parameters supplied by the user.

Another preferred method for determining the best overall match between the query nucleotide sequence (the sequence of the invention) and the subject sequence, also referred to as global sequence alignment, is based on the algorithm of Brutlag et al. (1990). It can be determined using the FASTDB computer program, the disclosure of which is incorporated herein by reference in its entirety. In a sequence alignment, both the query and subject sequences are DNA sequences. RNA sequences can be compared by first converting U to T. The results of the global sequence alignment are shown at the same rate. The preferred parameters used in the FASTDB alignment of DNA sequences to calculate percent identity are: matrix = unitary, k-set = 4, mismatch penalty = 1, joining penalty = 30, randomized base length = 0, cutoff score. = 1, Gap Penalty = 5, Gap Size Penalty 0.05, Window Size = 50
Either 0 or the length of the subject nucleotide sequence, which is 35 shorter. If the subject sequence is shorter than the query sequence due to 5'or 3'deletions rather than internal deletions, a manual correction to the results must be made. This is the FASTDB program
This is because 5 ′ and 3 ′ truncations of the target sequence are not taken into consideration when calculating the identity rate. For the query sequence, for the subject sequence truncated at the 5'or 3'end, the percent identity is 5'and 3'of the subject sequence that is not matched / aligned.
Is corrected by calculating the number of bases in the query sequence as a percentage of the total number of bases in the query sequence. Whether or not nucleotides are matched / aligned is determined by the result of FASTDB sequence alignment. This ratio is then subtracted from the percent identity calculated by the FASTDB program above using 10 as the specific parameter to obtain the final percent identity score. This corrected score is what is used for the purposes of the present invention. Only the nucleotides outside the 5'and 3'nucleotides of the subject sequence that do not match / align with the query sequence, as presented by the FASTDB alignment, are
Calculated for the purpose of manually adjusting the percent identity. For example, a 90 nucleotide subject sequence is aligned with a 100 nucleotide query sequence to determine percent identity. A deletion occurs at the 5'end of the subject sequence and therefore the FASTDB alignment does not show a match / alignment of the first 10 nucleotides at the 5'end. The 10 unpaired nucleotides represent 10% of the sequence (number of nucleotides at the 5'and 3'ends that do not match / total number of nucleotides in the query sequence) and therefore 10% from the identity score calculated by the FASTDB program. % Is subtracted. If the remaining 90 nucleotides were a perfect match, the final identity is 90%. In another example, a 90 nucleotide subject sequence is compared to a 100 nucleotide query sequence. In this case, because the deletion is an internal deletion, the nucleotides 5'or 3'of the subject sequence will match / align with the query sequence. In this case, the identity rate calculated by FASTDB is not manually corrected. That is, only the 5'and 3'nucleotides of the subject sequence that do not match / align with the query sequence are manually corrected. No other manual corrections are made for the purposes of the present invention.

Another preferred method for determining the best overall match between a query amino acid sequence (the sequence of the invention) and a subject sequence, also referred to as global sequence alignment, is based on the algorithm of Brutlag et al. (1990). Determined using the FASTDB computer program. In a sequence alignment, both the query and subject sequences are amino acid sequences. The results of the global sequence alignment are shown at the same rate. The preferred parameters used in the FASTDB amino acid alignment are: Matrix = PAM0, k-set = 2, mismatch penalty = 1, joining penalty = 20, randomized 25 base length = 0, cutoff score = 1, gap penalty = 5, Gap size penalty = 0.05, window size = 500 or the length of the target amino acid sequence, whichever is shorter. If the sequence of interest is shorter than the query sequence, due to N- or C-terminal deletions rather than internal deletions, results at the same rate must be manually corrected. This is because the FASTDB program does not take into account N- and C-terminal truncations of the subject sequence when calculating global identity. For a subject sequence that is truncated at the N- or C-terminus with respect to the query sequence, the percent identity is the residue of the query sequence that is the N- and C-terminus of the subject sequence that does not match / align with the corresponding subject residue. The number is corrected by calculating the number as a percentage of the total number of residues in the query sequence. Whether the residues are matched / aligned depends on FA
It is judged by the result of STDB sequence alignment. This ratio is then subtracted from the percent identity calculated by the FASTDB program above using the specified parameters to obtain the final percent identity score. This final identity score is what is used for the purposes of the present invention. Only N- and C-terminal residues of the subject sequence that do not match / align with the query sequence are considered for the purpose of manually adjusting the percent identity. It is only the interrogating amino acid residues outside the extreme N- and C-terminal residues of the subject sequence. For example, 90
The amino acid residue subject sequence is aligned with the 100 residue query sequence to determine percent identity. A deletion occurs at the N-terminus of the subject sequence, so the FASTDB alignment does not match / align with the first residue at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of unmatched N- and C-terminal residues / total number of residues in the query sequence), and thus the percent identity calculated by the FASTDB program. 10% is subtracted from the score. If the remaining 90 residues were perfectly matched, the final identity would be 90%. In another example, a 90 residue subject sequence is compared to a 100 residue query sequence. in this case,
Since the deletion is an internal deletion, residues on the N- or C-of the subject sequence will be matched / aligned with the query sequence. In this case, the identity rate calculated by FASTDB is not manually corrected. That is, only residue positions outside the N- and C-termini of the subject sequence that are not matched / aligned with the query sequence, as displayed by the FASTDB alignment, are manually corrected. No other manual corrections are made for the purposes of the present invention.

By “percent sequence similarity” is meant a comparison between polypeptide sequences, measured by comparing two sequences that are optimally aligned on a comparison window, where the polys within the comparison window. Peptide sequence portions may contain additions or deletions (ie gaps) when compared to a reference sequence (without additions or deletions) for optimal alignment between the two sequences. This percentage measures the number of positions that produce identical or equivalent amino acid residues in both sequences to yield the number of match positions, and divides the number of match positions by the total number of positions in the comparison window to give the percent sequence similarity. Calculated by multiplying the result by 100 to obtain.
Similarity is assessed using any of the various sequence comparison algorithms and programs known in the art, including those described above in this section. Equivalent amino acid residues are defined in the "Mutant Polypeptides" section herein. Polynucleotides of the Invention The present invention relates to GENSET genomic and cDNA sequences. The present invention is based on the GENSET gene, GE
Polynucleotides comprising NSET genomic and cDNA sequences, as well as fragments and variants thereof. These polynucleotides can be in purified, isolated, or recombinant form. The invention also includes allelic variants, orthologs, splice variants, and / or cognate species of the GENSET gene. Using methods known in the art and using the sequences disclosed herein or information from the clone pool deposited in the ATCC, full-length genes and cDNAs, allelic variants, splice variants, full-length coding portions. , Orthologs, and / or homologous species of genes and cDNAs corresponding to a nucleotide sequence selected from the group consisting of the sequences SEQ ID NOs: 1-241 and the sequences of clone inserts of the deposited clone pool. For example,
Allelic variants, orthologs and / or cognate species are those described in the section entitled "Searching for Similar Sequences" to generate suitable probes or primers from the sequences provided herein. Can be isolated and identified by screening for a suitable source of nucleic acid for the allelic variant and / or the desired homologue, using any of the techniques known to those of skill in the art.

In certain embodiments, the polynucleotide of the invention comprises at least 15, 30, 50,
It is 100, 125, 500 or 1000 consecutive nucleotides. In other embodiments, the polynucleotide has a length of 300 kb, 200 kb, 100 kb, 50 kb, 10 kb, 7.5 kb, 5 kb.
, 2.5kb, 2kb, 1.5kb, less than or equal to 1kb. In still other embodiments, the polynucleotides of the invention include portions of the coding sequence, as disclosed herein, but not all or part of the intron. In other embodiments, the polynucleotide comprising the coding sequence does not include the coding sequence of the genomic flanking gene (ie, 5'or 3'for the gene of interest in the genome). In other embodiments, the polynucleotides of the invention are 1000, 500, 250, 100, 75, 5
It does not include coding sequences for 0, 25, 20, 15, 10, 5, 5, 4, 3, 2, 1 or more naturally-occurring genomic flanking genes. Expression of the deposited clone pool GENSET gene of the present invention has been shown to induce the production of at least one mRNA species per GENSET gene, the cDNA sequence of which is published in the Appendix Sequence Listing as SEQ ID NOS: 1-241. CDNAs corresponding to these GENSET mRNA species (SEQ ID NOs: 1-241
) Was cloned into the vector pBluescriptII SK ⁻ (Stratagene) or one of its derivatives called pPT (see FIG. 1). Cells containing the cloned cDNA of the present invention are maintained as a permanent deposit by the inventors of Genset, SA, 24 Rue Royale, 75008 Paris, France. Table I shows the Applicant's internal designation number (column entitled "Internal Designation") assigned to each SEQ ID NO: 1 to 482 (column entitled "SEQ ID NO:"), where the sequence is a nucleic acid sequence or a polypeptide. Whether it is a sequence (column labeled "type") or a vector into which the cDNA was cloned (column labeled "vector"). Each cDNA is inserted into it by NotI Pst I double digestion.
If removed from the luescript vector and the cDNA sequence of interest does not contain this restriction site in its sequence, an appropriate fragment can be produced for each clone. pP
The preferred site of cDNA removal for those clones inserted in T is the desired c
If the DNA sequence does not contain this restriction site in its sequence, it is the site used for cloning, MunI and HindIII. On the other hand, other restriction enzymes at the vector's multiple cloning site can be used to recover the desired insert, as indicated by the manufacturer or as shown in FIG.

A pool of cells containing the cDNA of the invention, which makes it possible to obtain cells containing a particular polynucleotide, is also available in the American Culture Collection (ATCC), 10801 Universi.
Deposited at ty Boulevard, Manassas, VA 20110-2209, United States. Each cD
NA clones were transfected into separate bacterial cells (E. coli) for composite deposit. In particular, cells containing the sequences of SEQ ID NOs: 1-241 were deposited on January 21, 2000 as pool with ATCC accession number PTA-1218 and designated GENSET.07PRF. Bacterial cells containing a particular clone can be obtained from a composite deposit as follows. The oligonucleotide probe (s) should be designed for sequences known for the particular clone. This sequence may be derived from the sequences provided herein, or a combination of those sequences. The design of the oligonucleotide probe should preferably follow these parameters: (a) If there is a sequence region with the fewest ambiguous bases ("N"), that region should be designed. (B) Preferably, the probe is designed to have a melting point of about 80 ° C. (estimated at 2 ° C. for each A or T and 4 ° C. for each G or C). However, probes with melting points in the range of 40 ° C to 80 ° C may be used as long as specificity is not lost. Oligonucleotides should be labeled, preferably with gamma < ³² P> ATP (specific activity 6000 Ci / mol) and T4 polynucleotide kinase, using techniques commonly used for oligonucleotide labeling. Other labeling methods may be used. Unincorporated label should be removed by gel filtration chromatography or other established method. The amount of radioactivity incorporated into the probe should be quantified by counting with a scintillation counter. Preferably, the specific activity of the resulting probe should be about 4x10 ⁶ dpm / picomolar. Bacterial cultures containing pools of full length clones are preferably thawed to 100
(1 stock solution should be used to inoculate a sterile culture flask containing 25 ml sterile L-broth containing 100 (l / ml ampicillin. Cultures are preferably grown to saturation at 37 ° C. Saturated cultures should preferably be diluted with fresh L-broth Aliquots of these dilutions preferably contain 100 (l / ml ampicillin in a 150 mm Petri dish) Seeding on solid bacteriological medium containing L-broth and 1.5% agar to determine the dilution and volume that yields about 5000 distinct and distinct colonies when grown overnight at 37 ° C. Other known methods for obtaining clear and well-separated colonies may be used.

The colonies should then be transferred to a nitrocellulose filter, lysed, denatured and baked using standard colony hybridization methods. The filter is then 0.5% SDS, 100 pg / ml at 65 ° C for 1 hour with gentle agitation.
Yeast RNA, and 6X SSC (20 mM) containing 10 mM EDTA (approximately 10 ml / 150 mm filter)
X stock solution is 175.3 g NaCl / L, 88.2 g sodium citrate / L, pH 7.0 with NaOH.
Incubate in). Preferably, this probe is then added to 1x10 ⁶ dpm /
Add to hybridization mix at ml or higher concentration. The filters are then preferably incubated overnight at 65 ° C. for 1 hour with gentle agitation. The filters are then preferably washed in 500 ml of 2X SSC / 0.1% SDS for 15 minutes at room temperature with gentle shaking. As an option, a third wash with 0.1X SSC / 0.5% SDS may be performed for 30 minutes to 1 hour at 65 ° C. The filter is then preferably dried and autoradiographed for a time sufficient to observe the positive on X-ray film. Other known hybridization methods may also be used. Positive colonies are picked, cultivated, and plasmid DNA isolated using standard methods. Clones are then subjected to restriction analysis, hybridization analysis, or DNA
It can be confirmed by the sequencing method. Plasmid DNA obtained using these methods
May then be engineered using standard cloning techniques known to those of skill in the art. Alternatively, dD containing standard methods and primers designed at the multiple cloning site of the vector to recover the cDNA insert from the bacterial pool.
Isolated plasmid DNA using primers designed at both ends of NA insertion
PCR may be performed. For example, the PCR reaction can be performed with universal primers designed by the plasmid vendor or with primers specific for the cDNA of interest. In case of Bluescript SK (-), PCR reaction is sequence G
It can be carried out with a primer having GAAACAGCTATGACCA and a primer having the sequence GTAAAACGACGGCCAGT. This produces a DNA fragment containing the multiple clone site fragment and the cDNA insert. To recover a particular cDNA of interest, the sequence information obtained from the sequence listing in the Appendix can be used to design primers to be specific for the 5'and 3'ends of this cDNA. PC corresponding to the desired cDNA
The R product can then be manipulated using standard cloning techniques known to those of skill in the art.

Therefore, an object of the present invention is an isolated, purified or recombinant polynucleotide comprising a nucleotide sequence selected from the group consisting of cDNA inserts of the deposited clone pool. Furthermore, preferred polynucleotides of the invention consist of, consist essentially of or comprise a nucleotide sequence selected from the group consisting of the cDNA inserts of the deposited clone pools, purified, isolated, or recombinant GENSET cD.
Including NA. The polynucleotides of SEQ ID NOs: 1-141 may be compatible with the corresponding polynucleotides encoded by the human cDNA of the clone insert of the deposited clone pool. The polypeptides of SEQ ID NOS: 242-482 may be compatible with the corresponding polypeptides encoded by the human cDNA of the clone insert of the deposited clone pool. The correspondence between the polynucleotides of SEQ ID NOs: 1-141, the polypeptides of SEQ ID NOs: 242-482 and the clone inserts of the deposited clone pool is shown in Table I. CDNA sequence of the present invention Another object of the present invention is a purified, isolated, or recombinant polypolynucleotide comprising a nucleotide sequence selected from the group consisting of sequences SEQ ID NOs: 1-241, complementary sequences thereof, and fragments thereof. It is a nucleotide. Furthermore, preferred polynucleotides of the present invention include purified, isolated, or recombinant GENSET cDNAs that consist of, consist essentially of, or comprise sequences selected from the group consisting of SEQ ID NOs: 1-241. Next, an open reading frame capable of encoding a polypeptide is searched for the polynucleotide GENSET sequences of SEQ ID NOS: 1-241. GENSET
The ORF is also described in Von Heijne Nucleic Acids Re, whose publication is described in International Patent Application Publication WO 00/37491, the entire text of which is incorporated herein by reference.
s 14: 4683-4690, using a method slightly modified from the method disclosed in 1986,
Searched to identify potential signal sequence motifs. SEQ ID NOs: 242-272
And G of SEQ ID NOs: 1-31 and 33-143, which encode polypeptides of 274-283.
The ENSET cDNA was therefore found to contain such a signal peptide. The structural parameters of each cDNA of the present invention are set forth in Table II. That is, Table II sets forth SEQ ID NOS: 1-24 referenced by its SEQ ID NO (column entitled "SEQ ID NO").
1 of each cDNA, the positions of the first and last nucleotides of the coding sequence (listed under the heading "Complete coding sequence"), and, where applicable, of the secreted protein (SEQ ID NOS: 1-31 and 33-143). In this case, the position of the sequence encoding the signal sequence and the mature polypeptide, the position of the first and last nucleotides of the poly A signal ("" The "Poly A signal" is listed under the heading) as well as the first and last nucleotide positions of the poly A site (listed under the "Poly A site").

Thus, the complete coding sequence (CDS) or open reading frame (ORF) of each cDNA of the present invention means a nucleotide sequence that begins at the first nucleotide of the start codon and ends at the last nucleotide of the stop codon. (See the column entitled "Complete Coding Sequences" in Table II for sequences SEQ ID NOS: 1-241).
Similarly, the signal sequence of each cDNA of the invention begins with the first nucleotide of the start codon and ends with the last nucleotide of the codon encoding the signal peptide (SEQ ID NOS: 1-31 and 33-143 sequences). about,
(See the column entitled "Signal Sequence" in Table II) and for each cD of the invention.
The coding sequence for the mature polypeptide of NA is a nucleotide sequence that begins at the first nucleotide of the first codon to sequence and ends at the last nucleotide of the stop codon (see Tables for SEQ ID NOs: 1-31 and 33-143). II) (see column entitled "Matured Protein Coding Sequence"). Similarly, each cD of the present invention
The 5'untranslated region (or 5'UTR) of NA means a nucleotide sequence starting at nucleotide 1 and ending 5'adjacent nucleotides to the first nucleotide of the start codon. The 3'untranslated region (or 3'UTR) of each cDNA of the invention begins 3'adjacent nucleotides with respect to the last nucleotide of the stop codon, and c
By a nucleotide sequence ending at the last nucleotide of DNA. <Non-translated region> Furthermore, the present invention provides a nucleotide selected from the group consisting of the 5'UTR of the sequences of SEQ ID NOs: 1-241 and the sequence of the clone insert of the deposited clone pool, its complementary sequence, and its allelic variants. It relates to purified, isolated and recombinant nucleic acids containing sequences. The invention also comprises a nucleotide sequence selected from the group consisting of the 3'UTRs of sequences SEQ ID NOs: 1-241 and the clone inserts of the deposited clone pool, their complementary sequences, and allelic variants thereof, It relates to isolated and recombinant nucleic acids. These polynucleotides can be used to detect the presence of GENSET mRNA species in a biological sample using either hybridization or RT-PCR methods known to those of skill in the art. Furthermore, these polynucleotides are processed and matured of the polynucleotides containing them (either GENSET polynucleotides or heterologous polynucleotides), preferably the localization, stability and / or translation of said polynucleotides containing them. Can be used as a control molecule (see Decker and Parker, 1995, Derrigo et al., 2000 for UTR). Especially 3'UTR
The disclosure of which is incorporated by reference in its entirety into Makrides (19
99), US Pat. Nos. 5,925,56; 5,807,7 and 5,756,264, for modulating the stability of heterologous mRNA in a recombinant vector using any of the methods known to those of skill in the art. Can be used for

<Coding Sequence> Another object of the invention comprises the complete coding sequence of a sequence selected from the group consisting of the sequences SEQ ID NOs: 1-241, the clone inserts of the deposited clone pool, and variants thereof, It is an isolated, purified, or recombinant polynucleotide. A further object of the invention is an isolated, purified or recombinant polynucleotide encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOS: 242-482 and allelic variants thereof. Another object of the invention is to isolate, purify, or assemble a polypeptide comprising a sequence selected from the group consisting of the polypeptides encoded by the cDNA inserts of the deposited clone pool and allelic variants thereof. It is a modified polynucleotide. In a preferred embodiment, the invention includes an isolated, purified, or recombinant polynucleotide that encodes a polypeptide that comprises a sequence selected from the group consisting of the mature proteins of SEQ ID NOS: 242-272 and 274-384. To do. In another preferred embodiment, the invention provides an isolated, purified, or recombinant polynucleotide encoding a polypeptide comprising a sequence selected from the group consisting of the signal peptides of SEQ ID NOs: 242-272 and 274-384. Includes. As a result of sequencing errors, reverse transcription or amplification errors, mRNA splicing, post-translational modifications of the encoded protein, enzymatic cleavage of the encoded protein, or other biological factors, the size of the complete coding sequence may be different from that shown in the sequence listing in the Appendix. It will be appreciated that if clearly different, one of ordinary skill in the art can readily identify the size of the complete coding sequence in the sequence of SEQ ID NOs: 1-241. Therefore, SEQ ID NOS: 1-241
Claims relating to a nucleic acid comprising the complete coding sequence of
No readily recognizable changes or equivalents shall be excluded from the complete coding sequence described in the Sequence Listing in the Appendix. Similarly, when the size of the polypeptide is different from the size shown in the sequence listing in the Appendix as a result of any of the factors described above, the scope of claims for the polypeptide containing the amino acid sequences of the polypeptides of SEQ ID NOs: 242-482 is claimed. Does not exclude easily recognizable changes or equivalents from the sequences described in the Sequence Listing in the Appendix. As a result of sequencing errors, reverse transcription or amplification errors, mRNA splicing, post-translational modification of the encoded protein, enzymatic cleavage of the encoded protein, or other biological factors, the size of the mature protein coding sequence is shown in the Appendix Sequence Listing. It will be appreciated that one of ordinary skill in the art can readily identify the size of the mature protein coding sequence in the sequences of SEQ ID NOs: 1-31 and 33-143, if they differ significantly in size. Accordingly, the claims herein relating to a nucleic acid comprising the coding sequence of one mature protein of SEQ ID NOs: 1-33 and 33-143 are subject to readily recognizable variations from the coding sequences set forth in the Sequence Listing in the Appendix. Or equivalents will not be excluded. Similarly, if the mature polypeptide size is different from the size shown in the sequence listing in the Appendix as a result of any of the factors described above, then SEQ ID NOS: 242-272 and
Claims relating to mature polypeptides comprising the amino acid sequence of the 274-384 polypeptides are not intended to exclude readily discernible changes or equivalents from the sequences set forth in the Sequence Listing in the Appendix.

The size of the coding sequence of the signal peptide as a result of sequencing errors, reverse transcription or amplification errors, mRNA splicing, post-translational modification of the coding protein, enzymatic cleavage of the coding protein, or other biological factors. It will be appreciated that one of ordinary skill in the art can readily identify the size of the signal peptide coding sequence in the sequences of SEQ ID NOs: 1-31 and 33-143 if they are clearly different in size from those listed. Accordingly, the claims herein of a nucleic acid containing one signal sequence, SEQ ID NOs: 1-33 and 33-143, include readily recognizable changes or equivalents from the coding sequences set forth in the Sequence Listing in the Appendix. Will not be excluded. Similarly, if the signal peptide size is different from the size shown in the sequence listing in the Appendix as a result of any of the factors mentioned above, the signal peptide comprising the amino acid sequences of the polypeptides of SEQ ID NOs: 242-272 and 274-384. The following claims do not exclude the readily recognizable changes or equivalents from the entire coding sequence set forth in the Sequence Listing in the Appendix. The polynucleotides disclosed above comprising the coding sequence of the GENSET gene (full length protein of the mature protein) are expressed in the desired host cell or desired host organism when the polynucleotides are placed under the control of appropriate expression signals. Can be done. The expression signal can be either an expression signal contained in the control region present in the GENSET gene of the present invention or, in contrast, the signal is an exogenous regulatory nuclear sequence. Such polynucleotides, when placed under the appropriate expression signals, can be inserted into a vector for expression and / or amplification. The invention further includes polynucleotides that encode the polypeptides of the invention fused in frame to coding sequences for other heterologous amino acid sequences. Polynucleotides containing the GENSET signal sequence fused to a heterologous polypeptide as described in the section entitled "Secretion vectors" are of special interest. The invention includes polypeptides of the invention and additional non-coding, including, but not limited to, non-coding 5'and 3'sequences, vector sequences, sequences used for purification, probing, or priming. Nucleic acids that together encode the coding sequences are also included. For example, heterologous sequences include transcribed, non-translated sequences that may play a role in transcription, and mRNA processing, such as, for example, ribosome binding and mRNA stability. The heterologous sequence may alternatively include additional coding sequences that provide other functionality.
Thus, the nucleotide sequence encoding the polypeptide may be fused to a tag sequence, such as a peptide-encoding sequence that facilitates purification of the fusion peptide. In one preferred embodiment of this aspect of the invention, the tag amino acid sequence is a hexahistidine peptide, such as the tag provided in the pQE vector (QIAGEN), among many commercially available. For example, hexahistidine provides a convenient purification method for fusion proteins (see Gentz et al., 1989), the disclosure of which is incorporated herein by reference in its entirety. The "HA" tag is another peptide useful in purification that corresponds to an epitope derived from the influenza hemagglutinin protein (see Wilson et al., 1984), the disclosure of which is incorporated herein by reference in its entirety. ing.
As explained below, other fusion proteins, including the GENSET protein, are
Alternatively, it is fused to Fc at the C-terminus.

Suitable recombinant vectors containing a polynucleotide as described herein are disclosed elsewhere herein. Expression vectors encoding GENSET polypeptides or fragments thereof are described in the section entitled "Methods for Preparing Peptides." Regulatory Sequences of the Invention As mentioned above, the genomic sequence of the GENSET gene contains regulatory sequences in the non-coding 5'flanking regions and is the non-coding 3'flanking region of the GENSET coding regions containing the exons of these genes. The ranking region probably also contains control sequences. Polynucleotides from the GENSET 5'and 3'regulatory regions are useful for detecting the presence of at least one copy of the GENSET gene genomic nucleotide sequence or a fragment thereof in a test sample. <Preferred control sequences> Polynucleotides carrying control elements located at the 5'and 3'ends of the GENSET coding region can be conveniently used to control the transcriptional and translational activity of a heterologous polynucleotide of interest. Accordingly, the invention also relates to a purified or isolated nucleic acid comprising a polynucleotide selected from the group consisting of 5'and 3'terminal GENSET control regions, their complementary sequences, regulatory active fragments and variants thereof. The invention also conveniently has 99% nucleotide identity, preferably 99% nucleotide identity, with a polynucleotide selected from the group consisting of GENSET 5'and 3'regulatory regions, their complementary sequences, variants thereof and regulatory active fragments.
Purification comprising a polynucleotide having at least 95% nucleotide identity with a polynucleotide selected from the group consisting of GENSET 5 ′ and 3 ′ control regions, which is 99.5% nucleotide identity, and most preferably 99.9% nucleotide identity, or It relates to an isolated nucleic acid. Another object of the invention is the group consisting of the nucleotide sequences of the GENSET 5'and 3'regulatory regions, their complementary sequences, variants and regulatory active fragments thereof under stringent hybridization conditions as defined herein. A purified, isolated or recombinant nucleic acid containing a polynucleotide which hybridizes with a polynucleotide selected from the group consisting of polynucleotides selected from

Preferred fragments of the 5'regulatory region have a length of about 1500 or 1000 nucleotides, preferably about 500 nucleotides, more preferably about 400 nucleotides, more preferably 300 nucleotides, and most preferably 200 nucleotides. . Preferred fragments of the 3'regulatory region are at least 20, 50, 100, 150 in length.
, 200, 300 or 400 bases. For example, "providing" with respect to a biological sample, cell population, etc., indicates that the sample, cell population, etc., is in any way used in a method or procedure. Clearly, "providing" a biological sample or cell population does not require that the sample or cells be specifically isolated or obtained for the purposes of the present invention; instead, for example, by another individual, It may mean the use of the biological sample obtained for the purpose of A "regulatory active" polynucleotide derivative of the 5'regulatory region is a polynucleotide that is functional as a control region for expressing a recombinant polypeptide or recombinant polynucleotide in a recombinant cell host. A polynucleotide which comprises or consists of a fragment of said polypeptide. It can act as an enhancer or repressor. For purposes of the present invention, if the control polynucleotides described above include nucleotide sequences that contain transcriptional and translational control information, and such sequences are referred to as the desired polypeptide or the nucleotide sequence encoding the desired polynucleotide, " Nucleic acid or polynucleotide, when "operably linked," is "functional" as a control region for recombinant polypeptide or recombinant polynucleotide expression. The control polynucleotide of the present invention can be prepared from a nucleotide sequence which is a GENSET genome or cDNA sequence, for example, by cleavage using an appropriate control enzyme, or PCR. Regulatory polynucleotides can also be prepared by digestion of the GENSET gene containing genomic clones with exonuclease enzymes such as Bal31 (Wabi
Ko et al., 1986), the entire disclosure of which is incorporated herein by reference. These regulatory polynucleotides can also be prepared by chemical synthesis of nucleic acids, as described elsewhere herein. The control polynucleotide according to the present invention may be part of a recombinant expression vector that may be used to express the complete coding sequence in the desired host cell or host organism. Recombinant expression vectors according to the present invention are described elsewhere herein.

Preferred 5'regulatory polynucleotides of the present invention include the 5'-UTR of GENSET cDNA, or a regulatory active fragment or variant thereof. A more preferred 5'regulatory polynucleotide of the present invention has a sequence selected from the group consisting of 5'UTR of sequences 1-241, 5'UTR of clone inserts of deposited clone pools, regulatory active fragments and variants thereof. Including. Preferred 3'regulatory polynucleotides of the invention include the 3'-UTR of GENSET cDNA, or a regulatory active fragment or variant thereof. A more preferred 3'regulatory polynucleotide of the present invention has a sequence selected from the group consisting of 3'UTRs of sequences 1-241, 3'UTRs of clone inserts of the deposited clone pool, regulatory active fragments and variants thereof. Including. A further object of the invention consists of a purified or isolated nucleic acid comprising: a) (i) a GENSET 5'regulatory region or a nucleotide sequence comprising a polynucleotide which is the complement thereof; (ii) a GENSET 5'regulatory region or A nucleotide sequence comprising a polynucleotide having at least 95% nucleotide identity with its complementary polynucleotide; (iii) a GENSET 5'regulatory region or its complementary nucleotide sequence under stringent hybridization conditions A nucleotide sequence comprising a hybridizing polynucleotide, a polynucleotide comprising a 5'regulatory nucleotide sequence selected from the group consisting of; and (iv) (i), (ii), and (iii) the regulatory active fragment of the polynucleotide Or a variant; b) the desired polypeptide or nucleic acid molecule of interest. Is a nucleic acid molecule encoding
Said nucleic acid molecule is operably linked to a polynucleotide as defined in (a); and c) optionally comprising a 3'regulatory polynucleotide, preferably a 3'regulatory polynucleotide of the GENSET gene. In a particular embodiment, the nucleic acid as defined above comprises the 5'-UTR of GENSET cDNA or a regulatory active fragment or variant thereof. In a second particular embodiment, the nucleic acid as defined above is the 3'-UTR of the GENSET cDNA.
Or a regulatory active fragment or variant thereof.

The regulatory polynucleotide of the 5'regulatory region, or a regulatory active fragment or variant thereof, is operably linked to the 5'end of the nucleic acid molecule encoding the desired polypeptide or nucleic acid molecule of interest. The regulatory polynucleotide of the 3'regulatory region, or a regulatory active fragment or variant thereof, is operably linked to the 3'end of the nucleic acid molecule encoding the desired polypeptide or nucleic acid molecule of interest. The desired polypeptides encoded by the above nucleic acids may be of various nature or origin, including proteins of prokaryotic virus or eukaryotic origin. GENS
Polypeptides expressed under the control of the ET regulatory region include bacterial, fungal or viral antigens. In addition, eukaryotic proteins that are intracellular proteins such as "housekeeping" proteins, membrane-bound proteins such as mitochondrial membrane-bound proteins and cell surface receptors, and secreted proteins that are endogenous mediators such as cytokines. Included. The desired polypeptide is
It may be a heterologous polypeptide or a GENSET protein, especially SEQ ID NOS: 242-4
It may be a protein having an amino acid sequence selected from the group consisting of 82 sequences, fragments and variants thereof. The desired nucleic acid encoded by the above polynucleotides, which is usually an RNA molecule, is complementary to the desired coding polynucleotide, eg, the GENSET coding sequence, and thus may be useful as an antisense polynucleotide. Such polynucleotides may be included in a recombinant expression vector to express the desired polypeptide or nucleic acid in a host cell or host organism. Suitable recombinant vectors containing a polynucleotide as described herein are disclosed elsewhere herein. A cell is said to be "recombinant" with respect to a polynucleotide when the polynucleotide sequence is introduced into the host cell in a recombinant manner. Polynucleotide Variants The present invention relates to variants of the polynucleotides and fragments thereof described herein. As used herein, a "variant" of a polynucleotide is a polynucleotide that differs from a reference polynucleotide. In general, the differences are limited so that the reference nucleotide sequence and the variant are grossly similar and identical in many regions. The present invention includes both allelic variants and degenerate variants.

Examples of variant sequences of the polynucleotide of the invention are given in the sequence listing in the appendix. Table II
I lists all the similar sequences in the sequence listing, ie the SEQ ID NO of the variant. All cDNAs referenced by SEQ ID NO: on the given line in the table are considered to be variants of the same GENSET gene. <Allelic variant> The variant of the polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or a variant not known to occur naturally. By "allelic variant" is meant one of several alternative forms of a gene that occupies a given locus on the chromosome of an organism (see Lewin, 1990), the disclosure of which is herein incorporated by reference in its entirety. Incorporated as a reference. Diploid organisms can be homozygous or heterozygous for alleles. A non-naturally occurring variant of a polynucleotide is
Mutagenesis can be accomplished by techniques known in the art, including those applied to polynucleotides, cells or organisms. Degenerate Variants In addition to the isolated polynucleotides and fragments thereof of the present invention, the present invention further differs from those described above, but due to the degeneracy of the genetic code, the GENSET of the present invention. It includes a polynucleotide that includes a sequence that encodes a polypeptide. These polynucleotide variants are referred to as "degenerate variants" throughout this application. This completes all possible polynucleotide sequences encoding the GENSET polypeptides of the present invention. This includes the genetic code and species-specific codon selections known in the art. Thus, it is routine for one of ordinary skill in the art to produce the degenerate variants described above, eg, to optimize codon expression in a particular host (e.g., codons in human mRNA are Convert to the codon preferred by the bacterial host cell). The nucleotide changes present in the variant polynucleotide may be silent, which means that the amino acid encoded by the polynucleotide is not modified. However, nucleotide changes also result in amino acid substitutions in the polypeptide encoded by the reference sequence,
May cause additions, deletions, fusions and truncations. The substitutions, deletions or additions may be linked to one or more nucleotides. Variants may be modified in coding or non-coding regions, or both.
Modifications in the coding region may result in conservative or non-conservative amino acid substitutions, deletions or additions. In the context of the present invention, a preferred embodiment is one in which the polynucleotide variant encodes a polypeptide which possesses substantially the same biological properties or activities as the GENSET protein. More preferred polynucleotide variants are those that contain conservative substitutions.

Similar Polynucleotides Another embodiment of the present invention is a polynucleotide selected from the group consisting of SEQ ID NOs: 1-241 and the sequences of clone inserts of the deposited clone pool, and at least 90%, 95%, 96. A purified, isolated or recombinant polynucleotide which is%, 97%, 98% or 99% identical. The polynucleotide is included regardless of whether it encodes a polypeptide having GENSET biological activity. This is because one of ordinary skill in the art is already aware of how to use a nucleic acid molecule, eg, as a hybridization probe or primer, even when the nucleic acid molecule does not encode an active polypeptide. Uses of the nucleic acid molecules of the present invention, which do not encode a polypeptide having GENSET activity, include, inter alia, isolation of GENSET genes or allelic variants from DNA libraries, and GENSET mRNA or DNA analysis by Northern blot or PCR analysis. GENSET mRNA in biological samples that may be included
Including detection of expression. The invention further comprises a polynucleotide selected from the group consisting of the sequences SEQ ID NOs: 1-241 and the sequences of clone inserts of the deposited clone pool, and at least
A polynucleotide having 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity, wherein said polynucleotide is actually GE
NSET encodes a polypeptide having biological activity. Of course, due to the degeneracy of the genetic code, a polynucleotide selected from the group consisting of the sequences SEQ ID NOs: 1-241 and the sequences of the clone inserts of the deposited clone pool, and at least 50%, 60%,
One of ordinary skill in the art will immediately recognize that a number of polynucleotides that are 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical encode bioactive polypeptides. Will do. Indeed, since all degenerate variants of these nucleotide sequences encode the same polypeptide, this would be apparent to one of skill in the art without the comparative assay described above. It is further recognized in the art that a significant number of such non-degenerate variant nucleic acid molecules will also encode biologically active polypeptides. This is because those of skill in the art are familiar with amino acid substitutions that have little or no effect on protein function (eg, substituting one aliphatic amino acid with a second aliphatic amino acid). This will be explained in more detail below. The reference nucleotide sequence of the present invention and at least, for example, a polynucleotide having a nucleotide sequence that is 95% "identity", the polynucleotide sequence is 5 or less for each 100 nucleotides of the reference nucleotide sequence encoding the GENSET polypeptide. The nucleotide sequence of a polynucleotide is intended to be identical to a reference sequence, except that it contains a point mutation of. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, no more than 5% of the nucleotides in the reference sequence are deleted, inserted, or replaced with other nucleotides. Good. The query sequence is either the entire sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-241 and the sequences of the clone inserts of the deposited clone pool, or the group described above, or a fragment identified as described herein. It may be an ORF (open reading frame) of the polynucleotide sequence selected from

Hybridization Polynucleotides In another aspect, the invention comprises any of the methods known to those of skill in the art, including the methods disclosed herein and particularly in the “Search for Similar Sequences” section. It is used to provide an isolated or purified nucleic acid molecule that comprises a polynucleotide that hybridizes under stringent conditions with any of the polynucleotides of the invention. Nucleic acid molecules that hybridize to the polynucleotides of the invention under low stringency hybridization conditions, preferably moderate or low stringency conditions as defined herein, are also contemplated. Such hybridizing polynucleotides have a length of at least 15, 18, 20, 23, 25, 28,
It can be 30, 35, 40, 50, 75, 100, 200, 300, 500, 1000 or 2000 nucleotides. Of particular interest are polypeptides of the invention and GENSET polypeptides, especially
A polynucleotide that hybridizes to any of the polynucleotides encoding a GENSET polypeptide that exhibits GENSET biological activity. Of course, a polynucleotide that hybridizes only to a poly A + sequence (such as the 3'end poly A + tract of the cDNA shown in the sequence listing) or a 5'complementary stretch of T (or U) residues is The nucleotides hybridize to any nucleic acid molecule, including poly (A) stretches or complements thereof,
Not included in the definition of "polynucleotide" (eg, in practice a double-stranded cDNA clone produced using oligo dT as a primer). Complementary Polynucleotides The invention further provides an isolated nucleic acid molecule having a nucleotide sequence that is perfectly complementary to any of the polynucleotides of the invention. The present invention includes SEQ ID NOS: 1-24
1. An isolated, purified, or recombinant polynucleotide having a nucleotide sequence that is complementary to a sequence selected from the group consisting of the sequence of 1, the clone insert of the deposited clone pool, and fragments thereof. Such isolated molecules, especially DNA molecules, are useful as probes for gene mapping and identification of GENSET mRNA in biological samples by, for example, PCR or Northern blot analysis.

Polynucleotide Fragments The present invention further relates to polynucleotides that encode portions or fragments of the nucleotide sequences described herein. Applications of the polynucleotide fragments of the invention include probes, primers, molecular weight markers and expression of the polypeptide fragments of the invention. The fragment is a] a sequence of SEQ ID NOS: 1-241, b] a genome GENSET sequence, c] a polynucleotide encoding a polypeptide selected from the group consisting of sequences of SEQ ID NOS: 242-482, d] a clone of the deposited clone pool. A sequence of the insert and a polynucleotide sequence encoding the polypeptide sequence encoded by the clone insert of the e] deposited clone pool. The invention specifically includes purified or isolated polynucleotides that comprise at least 8 contiguous bases of a polynucleotide of the invention. In one aspect of this embodiment, the polynucleotide is at least 10, 12, 15, 18, 20, 25, 28, 30, 35, 40 that is a polynucleotide of the invention.
, 50, 75, 100, 150, 200, 300, 400, 500, 800, 1000, 1500 or 2000 consecutive nucleotides. In addition to the preferred polynucleotide size described above, a more preferred polynucleotide subgenus comprises at least 8 nucleotides, where "at least 8" means
Defined as an integer between the integer 8 and the integer indicating the 3'maximum nucleotide position published in the sequence listing or elsewhere herein. Preferred polynucleotides of the present invention further include the polynucleotide fragments described above, which are further specified for the 5'and 3'positions and are at least 8 nucleotides in length. The 5'and 3'positions are represented by the position numbers published in the sequence listing in the Appendix.
For alleles, degeneracy and other variants, position 1 is defined as the 5'maximum nucleotide of the ORF, ie, nucleotide "A" of the start codon where the remaining nucleotides are consecutive. Thus, all combinations of 5'and 3'nucleotide positions that a polynucleotide fragment of the invention may occupy on a polynucleotide of the invention that is at least 8 contiguous nucleotides in length are included in the invention as a separate species. . The polynucleotide fragments identified by the 5'and 3'positions are not listed individually, in order to be observed contiguously, and thus not to unnecessarily lengthen the present specification.

It is noted that the above species of polynucleotide fragments of the invention may alternatively be described by the formulas "ab", wherein "a" is the 5'maximum nucleotide position of the polynucleotide and "b" is 3 '. Equal to the maximum nucleotide position, and
a "is equal to an integer between 1 and the number of nucleotides of the polynucleotide sequence of the present invention minus 8, and" b "is an integer between 9 and the number of nucleotides of the polynucleotide sequence of the present invention. Equal; Further, "a" is at least 8 less than "b". The invention also provides for species exclusion of the polynucleotide fragments of the invention, identified by a subgenus of polynucleotides sized at the 5'and 3'positions or the nucleotides above. 5'and 3'as explained above
All fragments specified by position or nucleotide size can be excluded. The fragments described in Table IV are specifically excluded from the present invention. For these cDNAs, referred to by SEQ ID NO: Table IV shows the polyadenylation tail, and Al
Repetitive sequences containing u, L1, THE and MER repeats, SSTR sequences or satellites, microsatellites, and exclusion fragment positions in these sequence fragments with substantial homology to telomere repeats are indicated. Each fragment is designated by ab, where a and b are the start and end positions, respectively, of a given exclusion fragment. Excluded fragments are separated from each other by commas. As used herein, "polynucleotides described in Table IV" means in this manner
All polynucleotide fragments defined in IV are meant. Preferred inclusion and exclusion polynucleotide fragments of the invention are also listed in
It is described in Va and Table Vb. For these cDNAs referred to by SEQ ID NO: Tables Va and Vb show these sequences (columns entitled "fragments that are preferred to be included") and fragments that are preferably excluded ("fragments that are preferred to be excluded"). The position of the preferred fragment within the column) is indicated. Each fragment is designated by ab, where a and b are the start and end positions, respectively, of a given preferred fragment. The fragments are separated from each other by commas. As used herein, refer to Table V
By "excluded polynucleotides described in a and Vb" is meant all polynucleotides that are preferably excluded as described in Tables Va and Vb. As used herein, "preferred polynucleotides described in Tables Va and Vb" means all polynucleotide fragments, which are listed in Tables Va and Vb in this manner and which are preferably included.

Accordingly, the present invention is a sequence selected from the group consisting of the sequences SEQ ID NOs: 1-241 and the sequences that are completely complementary thereto, at least 8, 10, 12, 15, 18, 20
, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, 1000 or 2000 contiguous spans consisting of, consisting predominantly, or containing, isolated, purified, Or a recombinant polynucleotide, the flanking spans of these lengths being sized to match the length of the selected sequence, wherein said flanking spans are preferred polynucleotides described in Tables Va and Vb or At least 1, 2, 3, 5, 10, 15, 18, 20, 25, 28, 30, 35, 40, 50 of the sequences that are complementary
It contains 75, 100, 150, 200, 300, 400 or 500 nucleotides. The present invention is at least 8, 10, 12, 15, 18, 20, 25, 30, 35, which is a sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-241 and the sequences that are completely complementary thereto. 40, 50, 75
, 100, 150, 200, 300, 400, 500, 1000 or 2000 comprising an adjacent span consisting of, consisting of, or consisting essentially of, an isolated, purified, or recombinant polynucleotide, wherein , Said flanking spans comprise the preferred polynucleotides described in Tables Va and Vb, or sequences complementary thereto, the flanking spans of these lengths being sized to match the length of the selected sequence. The invention also includes an isolated span, comprising, consisting essentially of, or consisting of a contiguous span consisting of a polynucleotide selected from the group consisting of the sequences of SEQ ID NOs: 1-241 and the sequences that are fully complementary thereto. Or a recombinant polynucleotide,
Here, the flanking span comprises the preferred polynucleotides described in Tables Va and Vb, or sequences complementary thereto. Other preferred fragments of the invention are polynucleotides, including polynucleotides that encode a domain of the polypeptide. Such fragments can be used to obtain other polynucleotides encoding polypeptides having similar domains using hybridization or RT-PCR methods. Alternatively, these fragments may be used to express a polypeptide domain that may exhibit specific biological properties. Preferred domains for the GENSET polypeptides of the present invention are set forth in Table VI. Accordingly, another object of the present invention is the SEQ ID NO:
A sequence selected from the group consisting of 242-482 sequences, at least 5, 6, 8,
10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150, 200, 250,
An isolated, purified or recombinant polynucleotide encoding a polypeptide which consists of, consists essentially of, or consists of contiguous spans of 300, 350, 400, 450 or 500 contiguous amino acids, the length of which is Flanking spans are sized to match the length of the selected sequence, and the contiguous span comprises at least 1, 2, 3, 5, or 10 amino acid positions in the domain of the selected sequence. The present invention is also a sequence selected from the group consisting of the sequences of SEQ ID NOs: 242-482, at least 5, 6,
8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150, 200, 2
Includes isolated, purified or recombinant polynucleotides that encode a polypeptide comprising contiguous spans of 50, 300, 350, 400, 450 or 500 contiguous amino acids, contiguous spans of these lengths being selected by said selection. The size is matched with the length of the sequence, and the adjacent span is a domain of the selected sequence. The invention also provides SEQ ID NO:
Also included is an isolated, purified or recombinant polynucleotide that encodes a polypeptide comprising a sequence domain selected from the group consisting of sequences 242-482.

The present invention further includes combinations of any of the polynucleotide fragments listed in this section. Oligonucleotide Primers and Probes The invention also includes fragments of GENSET polynucleotides for use as primers and probes. Polynucleotides derived from GENSET genomic and cDNA sequences are useful for detecting the presence of at least one copy of GENSET polynucleotide or a fragment, complement or variant thereof in a test sample. <Structure Definition> The polynucleotide of the present invention can be used as a primer or a probe. Particularly preferred probes and primers of the invention are sequences selected from the group consisting of GENSET genomic sequences, cDNA sequences, and sequences that are perfectly complementary thereto.
At least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,
Includes isolated, purified, or recombinant polynucleotides that include contiguous spans of 200, 500, 1000, 1500 or 2000 nucleotides. Another object of the invention is selected from the group consisting of the sequences SEQ ID NOs: 1-241, the sequences of clone inserts of the deposited clone pool, the sequences which are perfectly complementary thereto, allelic variants thereof and fragments thereof. A purified, isolated, or recombinant polynucleotide that comprises a nucleotide sequence that is a sequence. Furthermore, preferred probes and primers of the invention consist of, consist essentially of or comprise the sequence of SEQ ID NOs: 1-241 and the sequence of the clone insert of the deposited clone pool, purified, isolated or recombinant GENSET cDNA. including. Particularly preferred probes and primers of the present invention are at least 12,15,18,20,25,30, which are sequences selected from the group consisting of sequences SEQ ID NOS: 1-241, and sequences which are perfectly complementary thereto. , 35, 40, 50,
Includes isolated, purified, or recombinant polynucleotides that include contiguous spans of 60, 70, 80, 90, 100, 150, 200, 500, 1000, 1500 or 2000 nucleotides. <Design of primer and probe> The probe or primer according to the present invention has a length in the range of 8 to 1000 nucleotides, and the length is at least 12, 15, 18, 20, 25, 35, 40, 50, 60. , 70, 80,
It has been specified to be 100, 250, 500, 1000, 1500 or 2000 nucleotides. More specifically, the lengths of these probes and primers are 8, 10, 15, 2
It can range from 0, or 30 to 100 nucleotides, preferably 10 to 50, more preferably 15 to 30 nucleotides. Short probes and primers tend to lack specificity for target nucleic acid sequences and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. The production of longer probes and primers is costly and often self-hybridizes to form hairpin structures. One of ordinary skill in the art can empirically determine the appropriate lengths of primers and probes under a particular set of assay conditions. The formation of stable hybrids depends on the melting temperature (Tm) of DNA. The Tm depends on the length of the primer or probe, the ionic strength of the solution and the G + C content. The higher the G + C content of the primer or probe, the higher the melting point, because the G: C pair is supported by three hydrogen bonds, while the A: T pair has only two hydrogen bonds. The GC content in the probe of the present invention is generally in the range of 10-75%, preferably 35-60%, and more preferably 40-55%.

For amplification purposes, primer pairs with approximately the same Tm are preferred. The primer is
Based on the GC content and melting point of the oligonucleotide, the OSP software (Hilli
er and Green, 1991) (the disclosure of which is incorporated herein by reference in its entirety), or the octamer frequency imbalance method (Griffais et al., 1991).
Based on PC-Rare (http: // bioinformatics.weizmann.ac.il/software/PC-R
are / doc / manuel.html), the disclosure of which is incorporated herein by reference in its entirety. DNA amplification techniques are known to those of skill in the art. Amplification techniques that can be used in connection with the present invention are EP-A-320 308, WO 9320227 and EP-A-
439 182, ligase chain reaction (LCR), polymerase chain reaction (PCR, RT-
PCR), and techniques such as nucleic acid sequence-based amplification described by Guatelli et al. (1990) and Compton (1991), Q-beta amplification as described in European Patent Application 4544610, Walker et al. 1996) and EP A 684 315, including, but not limited to, the strand displacement amplification method, the target-mediated amplification method described in International Patent Application Publication No. WO 9322461. The disclosures and disclosures thereof are incorporated herein by reference in their entireties. LCR and gap LCR are exponential amplification techniques, both based on DNA ligase for binding adjacent primers that are annealed to a DNA molecule. In the ligase chain reaction (LCR), a probe pair containing two primary (first and second) and two secondary (third and fourth) probes was used, all in molar excess relative to the target. To be The first probe hybridizes to the first segment of the target strand, and the second probe hybridizes to the second segment of the target strand, the primary probes contacting each other in a 5'phosphate-3 'hydroxyl relationship and thus ligase So that the two probes covalently fuse or combine to form a fusion product.
The 1st and 2nd segments are adjacent. In addition, the third (secondary) probe is the first
A portion of the probe can hybridize, and a fourth (secondary) probe can hybridize with a portion of the second probe in a similar contact fashion. Of course, if the target is initially double stranded, the secondary probe will also hybridize with the target complement in the first step. Once the binding strand of the primary probe is once separated from the target strand, it can hybridize to the third and fourth probes and bind to form a complementary, secondary binding product. It is important to note that the binding product is functionally equivalent to either the target or its complement. By repeating the cycle of hybridization and ligation reaction, amplification of the target sequence is achieved. Methods for multiplex LCRs have also been described (WO 9320227), the publication of which is incorporated herein in its entirety by reference. Gap LCR (GLCR) is a version of LCR in which the probes are not contiguous and are separated by 2 or 3 bases.

For amplification of mRNA, reverse transcription of mRNA into cDNA followed by polymerase chain reaction
(RT-PCR); or using one enzyme for both steps as described in US Pat. No. 5,322,770, or asymmetric gap LCR (RT-PCR as described by Marshall et al. (1994). The use of AGLCR) is within the scope of the present invention, the disclosures and disclosures of which are incorporated herein by reference in their entireties. AGLCR is a variant of GLCR that allows amplification of RNA. PCR technology is the preferred amplification method used in the present invention. Various PCR methods are known to those of skill in the art. For a survey of PCR technology, see White (1997), Erlich.
(1992) and "PCR method and its application" (1991, Cold Spring Harbor Laborat
See the publication titled Ory Press). The entire disclosures of these disclosures are incorporated herein by reference. In each of these PCR methods, PCR primers on either side of the nucleic acid sequence to be amplified were appropriately prepared with dNTPs and a temperature stable polymerase such as Taq polymerase, Pfu polymerase, Tth polymerase or Vent polymerase. Added to the nucleic acid sample. The nucleic acids in the sample are denatured and the PCR primers specifically hybridize to complementary nucleic acid sequences in the sample. The hybridized primer extends. Then another cycle of denaturation, hybridization, and extension is started.
The cycle is repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites. PCR is further described in US Pat. Nos. 4,683,195; 4,683,202; and
It is also described in several patents, including 4,965,188, the disclosures of which are incorporated herein by reference in their entireties. <Preparation of Primers and Probes> Primers and probes can be prepared, for example, by cloning and restriction of appropriate sequences, and phosphodiester method by Narang et al. (1979), phosphodiester method by Brown et al. (1979), Beaucage et al. Can be prepared by any suitable method, including direct chemical synthesis by methods such as the diethyl phosphoramidite method according to (1981) and the solid support method described in EP 0 707 592, the disclosures of which are incorporated herein by reference in their entirety. Have been incorporated as Detection probes are generally peptide nucleic acids, eg published in International Patent Application WO 92/20702, US Pat. Nos. 5,185,444; 5,034,506 and 5,142,047.
Nucleic acid sequences such as morpholine analogs or unmodified nucleic acid analogs, the disclosures of which are incorporated herein by reference in their entireties. Additional dNTP
The probe may have to be "non-extendable" because the can not be added to the probe. Analogs are usually non-extendable, and nucleic acid probes can be made non-extendable by modifying the 3'end of the probe so that the hydroxyl group cannot participate in extension. For example, the 3'end of the probe can be functionalized with a capture or detection label, thereby consuming or otherwise blocking hydroxyl groups. Alternatively, the 3'hydroxyl group is simply cleaved, substituted or modified. U.S. patent application Ser. Transferred.

Labeling of the Probes Any of the polynucleotides of the present invention may be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means, if desired,
It can be labeled by incorporating a label known in the art. For example, useful labels include radioactive substances (including ³² P, ³⁵ S, ³ H, ¹²⁵ I), fluorescent dyes (5-bromodesoxyuridine, fluorescein, acetylaminofluorene, digoxigenin).
Or it contains biotin. Preferably, the polynucleotide is labeled at its 3'and 5'ends. An example of non-radioactive labeling of nucleic acid fragments is given in French patent FR-78109.
75 or Urdea et al. (1988) or Sanchez-Pescador et al. (1988), the disclosures and disclosures of which are incorporated herein by reference in their entireties.
Furthermore, the probe according to the invention has structural features that allow signal amplification, such structural features are for example described in Urdea et al., 1991 or European patent E.
Branched DNA probes as described by P 0 225 807 (Chiron), the disclosures of which are incorporated herein by reference in their entireties. The detectable probe may be single-stranded or double-stranded, in vitro transcription,
It can be generated using techniques known in the art, including nick translation, or kinase reactions. A nucleic acid sample containing a sequence capable of hybridizing with the labeled probe is contacted with the labeled probe. If the nucleic acid in the sample is double-stranded, it may be denatured before contact with the probe. In some applications, nucleic acid samples may be immobilized on surfaces such as nitrocellulose or nylon membranes. Nucleic acid samples are genomic DNA, cDNA
It may include nucleic acids obtained from a variety of sources, including libraries, RNA, or tissue samples. The methods used to detect the presence of nucleic acid capable of hybridizing to a detectable probe include known techniques such as Southern blotting, Northern blotting, dot blotting, colony hybridization, and plaque hybridization. In some applications, a nucleic acid capable of hybridizing to a labeled probe can be cloned into a vector such as an expression vector, a sequencing vector, or an in vitro transcription vector to facilitate characterization and expression of the hybridizing nucleic acid in a sample. . For example, such techniques can be used to isolate and clone sequences in genomic or cDNA libraries that are capable of hybridizing to the detectable probes described herein.

Probe Immobilization Labels can also be used to capture primers to facilitate immobilization of either the primer or a primer extension product such as amplified DNA. The capture label is attached to the primer or probe and can be a specific binding member that forms a binding pair with the specific binding member of the solid phase reagent. Therefore, based on the type of label carried by the polynucleotide or probe,
It can be used to capture or detect target DNA. Further, it will be appreciated that the polynucleotides, primers or probes provided herein, themselves serve as capture labels. For example, if the binding member of the solid phase reagent is a nucleic acid sequence, it can be selected to bind to the complementary portion of the primer or probe, thereby immobilizing the primer or probe to the solid phase. If the polynucleotide probe itself serves as a binding member, those of skill in the art will recognize that the probe contains sequences that are not complementary to the label, also referred to as the "tail." If the polynucleotide primer itself serves as a capture label, at least a portion of the primer will be free to hybridize to the nucleic acid on the solid phase. DNA labeling techniques are known to those of skill in the art. The probes of the present invention are useful for many purposes. They are genomic DNA
In particular, it can be used in Southern hybridization with. The probe also
It can also be used to detect PCR amplification products. They also use other techniques, GENSET
It may be used to detect mismatches in genes or mRNA. Any of the polynucleotides, primers and probes of the invention can be conveniently immobilized on a solid support. The solid support is not critical and can be selected by one of ordinary skill in the art. Thus, latex particles, microparticles, magnetic beads, non-magnetic beads (including polystyrene beads), membranes (including nitrocellulose pieces), plastic tubes, microtiter well walls, glass or silicon chips, sheep (or other suitable) Red blood cells and duracytes are all suitable examples. Suitable methods for immobilizing nucleic acids on a solid phase include ionic, hydrophobic, covalent interactions and the like. As used herein, solid support means a material that is insoluble or can be made insoluble by a subsequent reaction. Solid supports can be chosen for their unique ability to attract and immobilize capture reagents. Alternatively, the solid phase can carry other receptors that have the ability to attract and immobilize capture reagents. The additional acceptor may comprise a charge material that is oppositely charged with respect to the capture reagent itself or with respect to the charge material that is bound to the capture reagent. Still further, the acceptor molecule can be a specific binding member that has the ability to be immobilized (attached) on a solid support and to immobilize the capture reagent by a specific binding reaction. The acceptor molecule allows for indirect attachment of the capture reagent to the solid support material before or during the assay. Solid phases are therefore plastics, derivatized plastics, magnetic or non-magnetic metals, glass or silicon surfaces of test tubes, microtiter wells, sheets, beads, microparticles, chips, sheep (or other suitable animal). It can be red blood cells, duracytes® and other shapes known to those skilled in the art. The polynucleotides of the invention are individually attached to or immobilized on a solid support, or at least 2, 5, 8, 10, 12, 15, 20, or 25.
As a group of different polynucleotides of the invention can be attached or immobilized on a single solid support. Furthermore, a polynucleotide other than the polynucleotide according to the invention may be attached to the same solid support as one or more of the polynucleotides of the invention.

<Oligonucleotide Array> A substrate containing a plurality of oligonucleotide primers or probes of the present invention is
Whether used for the detection or amplification of target sequences in the GENSET gene, and for the detection of mutations in the coding or non-coding sequences of the GENSET gene, and also processes (embryogenesis, disease treatment) Can be used to measure the expression of the GENSET gene in different contexts, such as different tissues at different stages, and in patients versus healthy individuals as described elsewhere in this application. As used herein, "array" means a one-dimensional, two-dimensional, or multidimensional array of nucleic acids of sufficient length to allow specific detection of gene expression. For example, the array may include multiple nucleic acids from the gene whose expression level is to be evaluated. The array comprises GENSET genomic DNA, GENSET cDNA, its complementary sequences or fragments thereof. Preferably, the fragment has a length of at least
It is 12, 15, 18, 20, 25, 30, 35, 40 or 50 nucleotides. More preferably, the fragment length is at least 100 nucleotides. Even more preferably, the length of the fragment is at least 100 nucleotides or more. In certain embodiments, the fragments can be 500 nucleotides or more in length. Any of the polynucleotides provided herein can be attached to overlapping regions or random locations on the solid support. Alternatively, the polynucleotides of the invention may be attached in an aligned array in which each polynucleotide is attached to a discrete region of the solid support that does not overlap with the attachment sites of the other polynucleotides. Preferably, such aligned arrays of polynucleotides are designed to be "addressable" in which individual locations are recorded and accessible as part of the assay. Addressable polynucleotide arrays generally include a plurality of different oligonucleotide probes conjugated to a substrate surface at different known locations. Knowledge of the exact location of each polynucleotide location makes these "addressable" arrays particularly useful in hybridization assays. Any of the addressable array technologies known in the art can be used with the polynucleotides of the invention. One embodiment of these polynucleotide arrays is known as Genechips® and is generally described in US Pat.
, 143,854, International Patent Application Publication Nos. WO 90/15070 and 92/10092, the disclosures of which are incorporated herein by reference in their entireties. These arrays are generally based on a photo-induced synthetic method (Fodor et al., 1) which combines mechanical synthesis or a combination of photolithography and solid phase oligonucleotide synthesis.
991), the disclosure of which is incorporated by reference in its entirety. Immobilization of oligonucleotide arrays on a solid support is commonly identified as "Very Large Immobilized Polymer Synthesis (VLSIPS®)," where the probes are immobilized as a dense array on the solid surface of the chip. This is made possible by the development of the technology described in VLSIPS® technology, an example of which is US Pat.
, 854; and 5,412,087, International Patent Application Publications WO 90/15070, WO 92/10092, WO 95/11995, the disclosures of which are incorporated herein by reference in their entirety. It describes methods for forming oligonucleotide arrays by techniques such as light-induced synthesis. Additional presentation strategies for aligning and displaying oligonucleotide arrays on a chip in an attempt to maximize hybridization pattern and sequence information in a design strategy aimed at providing immobilized nucleotide arrays on a solid support. Was developed. Examples of such presentation strategies are international patent application publications WO 94/12305, WO 94/11530, WO 97/29212 and WO 97/31256.
Issue, which is incorporated herein by reference in its entirety.

Finally, the invention relates to an array of nucleic acid molecules comprising at least one polynucleotide of the invention, in particular probes or primers as described herein. Preferably, the invention relates to an array of nucleic acid molecules comprising at least two polynucleotides of the invention, especially probes or primers as described herein. Preferably, the invention relates to an array of nucleic acid molecules comprising at least 5 polynucleotides of the invention, in particular probes or primers as described herein. A preferred embodiment of the present invention is at least 1,2,5,10,15 selected from the group consisting of the sequences of SEQ ID NOs: 1-241 and the sequence of the clone insert of the deposited clone pool, its complete complement sequence and fragments thereof, At least 12, 15, 18, 20, 25, 30, 35, 40, 50, 1 in length, containing 20, 35, 50, 100, 150 or 200 sequences
A polynucleotide array that is 00, 500, 1000, 1500, or 2000 nucleotides. Method of Producing Polynucleotide of the Present Invention The present invention also includes a method of producing the polynucleotide of the present invention, which comprises the polynucleotides of SEQ ID NOs: 1-241, the genomic DNA obtained therefrom, or a fragment thereof. These methods involve sequentially joining nucleotides together to produce a nucleic acid having the above sequence. Polynucleotides of the invention can be enzymatically or chemically synthesized using techniques known to those of skill in the art, including amplification or hybridization based methods as described herein. Various chemical methods for nucleic acid synthesis are known to those of skill in the art. In many of these methods, the synthesis is performed on a solid support. These include the 3'phosphoramidite method in which the 3'end groups of the desired oligonucleotide are immobilized on an insoluble carrier. The additional nucleotide group is blocked at the 5'hydroxyl group as well as activated at the 3'hydroxyl group to cause binding with the immobilized nucleotide group.
Deblocking the neoimmobilized nucleotide compound as well as repeating the cycle will produce the desired polynucleotide. Alternatively, the polynucleotide can be prepared according to US Pat.
It can be prepared as described in 049,656, the disclosure of which is incorporated herein by reference in its entirety. In certain embodiments, several polynucleotides prepared as described above are combined and combined to produce a longer polynucleotide having the desired sequence.

Polypeptides of the Invention As used herein, "GENSET polypeptide" refers to all of the proteins and polypeptides of the invention. The present invention consists of a sequence selected from the group consisting of SEQ ID NOs: 242 to 482, consisting of, predominantly comprising, or comprising a recombinant form thereof,
An isolated or purified GENSET polypeptide, a polypeptide encoded by a human cDNA contained in a deposited clone, a mature protein contained in SEQ ID NOS: 242-272 and 274-384, a mature protein encoded by a clone insert of a deposited clone pool, And GENSET polypeptides, including variants thereof. Another object of the invention is the polypeptides encoded by the polynucleotides of the invention, as well as fusion polypeptides comprising such polypeptides. Polypeptide Variants The present invention further provides GENSET polypeptides encoded by allelic and splice variants, orthologs, and / or cognate species. Of the group of polypeptides consisting of SEQ ID NOs: 242-482, the mature proteins contained in SEQ ID NOs: 242-272 and 274-384, and the polynucleotides encoded by the full-length or mature polypeptides encoded by the clone inserts in the deposited clone pool. Known in the art using the sequences disclosed herein or information from clones deposited with the ATCC to obtain allelic variants, splice variants, orthologs, and / or cognate species. You can use the method that is. The polypeptides of the present invention may also include a polypeptide selected from the group consisting of the sequences of SEQ ID NOS: 242-482, the mature protein contained in the sequences of SEQ ID NOS: 242-272 and 274-384, and cloned into the deposited clone pool. Encoded full length or mature polypeptide and at least 50%, more preferably at least
60%, and even more preferably 70%, 80%, 90%, 95%, 96%, 97%, 98%
, Or a polypeptide having an amino acid sequence that is 99% identical. A polypeptide having an amino acid sequence that is at least 95% “identical” to a query amino acid sequence of the invention means that the subject polypeptide sequence contains no more than 5 amino acid modifications for each 100 amino acids of the query amino acid sequence. Except for that, it means that the amino acid sequence of the subject polypeptide is identical to the query sequence. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the query amino acid sequence, no more than 5% (5 out of 100) amino acid residues in the subject sequence are inserted, deleted, ( Indel) or other amino acids.

Further polypeptides of the invention include polypeptides having at least 90%, more preferably 95%, and even more preferably 96%, 97%, 98% or 99% similarity to that described above. . A polypeptide having at least, for example, 95% "similar" amino acid sequence to a query amino acid sequence of the invention is that the subject polypeptide sequence comprises no more than 5 amino acid modifications for each 100 amino acids of the query amino acid sequence. Otherwise, it means that the amino acid sequence of the subject polypeptide is similar to the query sequence (ie includes the same or equivalent amino acid residues). In other words, in order to obtain a polypeptide having an amino acid sequence that is at least 95% similar to the query amino acid sequence, no more than 5% (5 out of 100) amino acid residues in the subject sequence are inserted, deleted, (indeled). ) Or other non-equivalent amino acids. Such modifications of the reference sequence can occur at the amino or carboxyl terminal positions of the reference amino acid sequence, or anywhere between those terminal positions, either separately between residues in the reference sequence or within the reference sequence. It may be interspersed as one or more adjacent groups. The query sequence can be the entire amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 242-482, and the entire amino acid sequence encoded by the clone insert of the deposited clone pool or a fragment identified herein. Variant polypeptides described herein are included in the invention regardless of whether they have normal biological activity. This is because one of skill in the art is already aware of how to use a polypeptide, eg, as a vaccine or to raise antibodies, even if the polypeptide molecule has no biological activity. GENSET
Other uses of the polypeptides of the present invention that have no biological activity include, inter alia, molecular weight as epitope tags in epitope mapping and on SDS-PAGE gels or molecular sieve gel filtration columns using methods known to those of skill in the art. Including markers. As described below, the polypeptides of the invention also produce polyclonal and monoclonal antibodies useful as assays for detection of GENSET protein expression or agonists and antagonists capable of enhancing or suppressing GENSET protein function. Can also be used for Furthermore, such polypeptides can be used in the yeast two-hybrid system to "capture" GENSET protein binding proteins that are also agonist and antagonist candidates according to the invention (see, eg, Fields et al. 1989). , The disclosure of which is incorporated herein by reference in its entirety.

Methods of Preparing the Polypeptides of the Invention The polypeptides of the invention can be prepared by any suitable method. Such polypeptides include isolated naturally derived polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. The polypeptides of the present invention are preferably supplied in isolated form and may be partially or preferably substantially purified. Finally, the invention also relates to the polypeptides of the invention, in particular the polypeptides encoded by the cDNAs of SEQ ID NOs: 1-241, the mature proteins encoded by the fragments of SEQ ID NOs: 1-31 and 33-143, of the deposited clone pool. Full-length and mature polypeptides encoded by clone inserts, genomic DNs derived therefrom
A, or a fragment thereof, and a method of making the polypeptides of SEQ ID NOS: 242-482, the mature polypeptides included in SEQ ID NOS: 242-272 and 274-384, or fragments thereof. These methods involve sequentially joining the amino acids together to produce a nuclear polypeptide having the above sequence. In certain embodiments, the polypeptides produced by these methods have a length of 15
It has 0 amino acids or less. In other embodiments, the polypeptides produced by these methods are 120 amino acids or less in length. <Isolation> [From Natural Source] The GENSET protein of the present invention can be isolated from a natural source, including human or non-human animal body fluids, tissues and cells (directly isolated cells or cultured cells). Methods for extracting and purifying native proteins are known in the art and include the use of detergents or chaotropic agents to disrupt the particles, followed by ion exchange chromatography, affinity chromatography, specific gravity precipitation, and gel electrophoresis of the polypeptide. Includes differential extraction and separation. See, for example, "Methods of Enzymology, Academic Press, 1993" for various methods for protein purification. The entire disclosure of which is incorporated herein by reference. Polypeptides of the invention may also be conjugated to antibodies of the invention, such as those described herein, by methods known in the protein purification arts,
It can also be purified from natural sources.

From Recombinant Sources Preferably, the GENSET polypeptides of the invention are recombinantly produced using common expression methods known in the art. The polynucleotide encoding the desired polypeptide is operably linked to a promoter in an expression vector appropriate to the accessible host. Both eukaryotic and prokaryotic host systems are used for recombinant polypeptide formation. The polypeptide is then isolated from the lysed cells or culture medium and purified to the extent necessary for the intended use. Any of the GENSET polynucleotides set forth in SEQ ID NOs: 1-241, the clone inserts of the deposited clone pool, and allelic variants thereof can be used to express the GENSET polypeptide. The nucleic acid encoding the GENSET polypeptide to be expressed is operably linked to the promoter in the expression vector using conventional cloning technology. GE in expression vector
The NSET insert may comprise the complete coding sequence of the GENSET protein or a part thereof, especially the sequence of the mature polypeptide. For example, the GENSET-derived insert is a GENSET protein selected from the group consisting of the sequences of SEQ ID NOs: 242-482 and the polypeptide encoded by the clone insert in the deposited clone pool, at least 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75
, A polypeptide containing 100, 150 or 200 contiguous amino acids may be encoded. Accordingly, a further embodiment of the invention is a method of producing a polypeptide comprising a protein selected from the group consisting of the sequences SEQ ID NOs: 242-482, and the polypeptide sequence encoded by the clone insert of the deposited clone pool. ,
The method comprises the following steps: a) i) sequences of SEQ ID NOs: 1-241, ii) sequences of clone inserts of the deposited clone pool, iii) sequences encoding one of the polypeptides of SEQ ID NOs: 242-482, And iv) obtaining a cDNA comprising a sequence selected from the group consisting of a polynucleotide sequence encoding a polypeptide encoded by one of the clone inserts of the deposited clone pool; b) the cDNA operably linked to a promoter. As such, inserting the cDNA into an expression vector; and c) introducing the expression vector into a host cell, whereby the host cell produces the polypeptide.

In one aspect of this embodiment, the method further comprises the step of isolating the polypeptide. Another embodiment of the invention is a polypeptide obtainable by the method described in the paragraph above. The expression vector is any of the mammalian, yeast, insect or bacterial expression systems known in the art. Commercially available vectors and expression systems are available at the Genetics Institute (Camb
ridge, MA), Stratagene (La Jolla, California), Promega (Madison, Wisconsi
n), and Invitrogen (San Diego, California). If desired, to enhance expression and to facilitate correct protein folding, as described in U.S. Pat.No. 5,082,767, the disclosure of which is incorporated herein by reference in its entirety. ,), The codon content and the codon pairs of the sequence are optimized for the particular expression organism into which the expression vector is introduced. In one embodiment, the entire coding sequence of the GENSET cDNA and the 3'UTR via the poly A signal of the cDNA are operably linked to the promoter in the expression vector.
Alternatively, if the nucleic acid encoding the portion of the GENSET protein lacks methionine, which serves as the initiation site, the initiation methionine can be introduced adjacent to the first codon of the nucleic acid using conventional techniques. Similarly, if the insert from the GENSET cDNA lacks the poly A signal, this sequence can be used, for example, in BglI and Sa
It can be added to the construct by splicing the polyA signal from pSG5 (Strategene) using the Il restriction endonuclease enzyme and incorporating it into the mammalian expression vector pXT1 (Stratagene). pXTl contains a portion of the gag gene from LTR and Moloney murine leukemia virus. The position of the LTR in the construct allows efficient stable transfection. The vector contains the herpes simplex thymidine kinase promoter and the selectable neomycin gene. A nucleic acid encoding the GENSET protein or a portion thereof is complementary to the GENSET cDNA or a portion thereof, and corresponds to the restriction endonuclease sequence for PstI and the cDNA 3'primer incorporated into the 5'primer 5; Clone inserts of SEQ ID NOs: 1-241 and the deposited clone pool, using oligonucleotide primers containing BglII at the ends, carefully checking that the sequence encoding the GENSET protein or a portion thereof is correctly positioned with respect to the poly A signal. Is obtained by PCR from a vector containing the GENSET cDNA selected from the group consisting of PCR
The purified fragment resulting from the reaction is digested with PstI, blunted with exonuclease, digested with BglII, purified and ligated into pXT1, thereby containing the poly A signal and further digested with BglII. .

Alternatively, the cDNA encoding the secreted protein is pED6dpc2 (DiscoverEase, Ge
clones in the netics Institute, Cambridge, MA). The resulting pED6dp
The c2 construct may be transfected into a suitable host cell such as COS1 cells. Methoxylate resistant cells are selected and expanded. Preferably, the secreted protein expressed from the cDNA is released into the culture medium, thereby facilitating purification. In other embodiments, recombinant polynucleotides encoding secretory or leader sequences, prosequences, sequences that aid purification, such as multiple histidine residues, or additional sequences for stability during recombinant production. It is often advantageous to add nucleotide-added nucleotide sequences. As a control, an expression vector lacking the cDNA insert is introduced into a host cell or organism. Transfection of the GENSET expression vector into mouse NTH 3T3 cells is just one embodiment of introducing the polynucleotide into the host cell. Insertion of a polynucleotide encoding a polypeptide into a host cell is affected by calcium phosphate transfection, DEAE dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Sometimes. Such methods are described in a number of standard laboratory manuals, such as Davis et al. (1986), the disclosure of which is incorporated herein by reference in its entirety. It is specifically contemplated that the polypeptides of the present invention may actually be expressed by host cells lacking the recombinant vector. If the recombinant cell extract, or expressed polypeptide, is secreted, the protein from the culture medium is then prepared and the proteins are separated by gel electrophoresis. If desired, the proteins may be precipitated or separated with ammonium sulfate based on size or charge prior to electrophoresis. The proteins present are detected using techniques such as Coomassie or silver staining or antibodies to the protein encoded by the GENSET cDNA of interest. Coomassie and silver staining techniques are known to those skilled in the art. A protein from a host cell or organism containing an expression vector containing a GENSET cDNA or fragment thereof is compared to a protein from a control cell or organism. The presence of bands from cells containing the expression vector that were not present in control cells was determined by GENS
Indicates that the ET cDNA is being expressed. In general, the band corresponding to the protein encoded by GENSET cDNA is mobile near the predicted amino acid number in the open reading frame of the cDNA. However, the band has a different mobility than that expected as a result of modifications such as glycosylation, ubiquitin binding, or enzymatic cleavage.

Alternatively, the GENSET polypeptide to be expressed may also be a product of a transgenic animal, ie a transgenic bovine, goat, porcine or pig characterized by somatic and germ cells containing a nucleotide sequence encoding the protein of interest. It can be a component of sheep milk. Polypeptides of the invention are known in the art including fractional extraction, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lecithin chromatography. Depending on the method, it can be recovered as well as purified from recombinant cell culture. For example, see “Enzymatic Methods” above for various methods for protein purification.
checking ... More preferably, high performance liquid chromatography (“HPLC”) is used for purification. Recombinantly produced GENSET polypeptides can be substantially purified using techniques described herein or otherwise known in the art. An example is the one-step method described in Smith and Johnson (1988), the disclosure of which is incorporated herein by reference in its entirety.
Polypeptides of the invention can also be purified from recombinant sources using antibodies directed against the polypeptides of the invention, such as those described herein, in a manner known in the protein purification arts. You can also do it. Preferably, the recombinantly expressed GENSET polypeptide is purified using standard immunochromatographic techniques, such as those described in the section entitled "Immunoaffinity Chromatography". In such methods, a solution containing the protein of interest, such as a culture medium or cell extract, is applied to a column having antibodies to the protein attached to a chromatography matrix. The recombinant protein is attached to the immunochromatographic column. The column is then washed to remove non-specifically bound proteins. The specific binding protein is then released from the column and further recovered using standard techniques. If antibody production is not possible, the GENSET cDNA sequence or fragments thereof can be incorporated into an expression vector designed for use in purification schemes using chimeric polypeptides. In such a strategy, GE
The coding sequence of the NSET cDNA or fragment thereof is inserted in frame with the genes encoding the other half of the chimera. The other half of the chimera may be a beta-globin or a nickel binding polypeptide encoding sequence. The chromatographic matrix with antibodies to beta-globin or nickel attached to it is then used for purification of the chimeric protein. Protease cleavage sites are beta-globin or nickel binding polypeptides and GENSET
It can be manipulated between cDNAs or fragments thereof. Thus, the two chimeric polypeptides can be separated from each other by protease digestion.

One useful expression vector for the production of beta-globin chimera is pSG5 (Stratagene) which encodes rabbit beta-globin. Intron II of the rabbit beta-globin gene facilitates splicing of expressed transcripts and the polyadenylation signal incorporated into the construct increases expression levels. These techniques as described are known to those skilled in the field of molecular biology. Standard methods are published as method textbooks such as Davis et al. (1986), and many methods are available.
Available from Stratagene, Life Technologies, Inc. or Promega. Other polypeptides include In Vitro Express® Translation Kit (Stratagen
In vitro translation systems such as e) can be used to produce from constructs. Depending on the host used in recombinant production, the polypeptides of the present invention may be glycosylated or non-glycosylated. In addition, the polypeptides of the present invention may also include an initial modified methionine residue, in some cases as a host-mediated process. Therefore, it is known that the N-terminal methionine encoded by the translation initiation codon is generally highly efficiently removed from any protein after translation in all eukaryotic cells. The N-terminal methionine found on most proteins is
Although efficiently removed in most prokaryotes, for some proteins this prokaryotic removal process is inefficient due to the nature of the amino acids to which the N-terminal methionine is covalently attached. [From chemical synthesis] Furthermore, the polypeptides of the present invention, particularly when they are short protein fragments, can be chemically synthesized using techniques known in the art (eg, Creighton, 1
983; and Hunkapiller et al., 1984), the disclosure of which is incorporated herein by reference in its entirety. For example, a polypeptide corresponding to a fragment of the polypeptide sequence of the present invention can be synthesized using a peptide synthesizer. Various methods of making polypeptides are known to those of skill in the art, including those in which the carboxyl terminus of an amino acid is attached to polyvinylbenzene or other suitable resin. The amino acid to be added contains a blocking group on the amino moiety and side chain reactive groups so that only the carboxyl moiety can react. Carboxyl groups are activated with arbodiimide or other activators, allowing them to be conjugated to immobilized amino acids. After removal of the blocking group, the cycle is repeated to produce a polypeptide with the desired sequence. Alternatively, the method described in US Pat. No. 5,049,656 may be used. The entire publication is incorporated herein by reference.

Furthermore, if desired, non-traditional amino acids or amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-traditional amino acids are
D-isomers of common amino acids, 2,4-diaminobutyric acid, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-
Aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acid , B-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and designer amino acids such as common amino acid analogs, but are not limited thereto. Further, the amino acid may be D (dextrorotatory) or L (levorotatory). <Modification> The present invention can be carried out by known protection / blocking groups, proteolytic cleavage, binding to antibody molecules or other cellular ligands, etc., for example, by glycosylation, acetylation, phosphorylation, amidation, derivatization, It includes polypeptides that are specifically modified during or after translation. Other post-translational modifications encompassed by the present invention include, for example, N-linked or O-linked carbohydrate chains, N- or C-terminal processing), attachment of compound moieties to amino acid backbones, N-linked or O-linked carbohydrate chain chemistry. Modifications and additions or deletions of N-terminal methionine residues as a result of prokaryotic host cell expression. The polypeptide may also be modified with a detectable label such as an enzyme, a fluorescent, an isotope or an affinity label to allow detection and isolation of the protein. The invention also provides derivatives of the polypeptides of the invention by chemical modification which confer other advantages such as solubility, improved polypeptide stability and circulation time, or reduced immunogenicity. See U.S. Pat. No. 4,179,337. The compound portion for derivatization can be selected with reference to U.S. Patent No. 4,179,337,
The entire publication is incorporated herein by reference. The compound moiety for derivatization can be selected from water soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol and the like. Polypeptides can be modified at random positions within the molecule or at predetermined positions within the molecule and can include one, two, three or more attachment compound moieties.

The molecular weight of the polymer is not limited, and it may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is in the range of about 1 kDa to about 100 kDa for ease of handling and manufacturing ("about" means that in the preparation of polyethylene glycol, the molecular weight varies above and below the stated molecular weight. Means that there is). The desired therapeutic profile (eg, desired duration of release, effect of biological activity, ease of handling, degree of antigenicity and lack thereof, and other known effects of polyethylene glycol on therapeutic proteins or analogs). Other sizes may be used based on The polyethylene glycol molecule (or other compound moiety) should be attached to the protein, taking into account its effect on the functional or antigenic domains of the protein. There are numerous attachment methods available to the person skilled in the art, for example EP 0 401 384, (PEG
Binding to GCSF), and Malik et al. (1992) (of GM-CSF using tresyl chloride).
PEGylation), the entire disclosure of which is incorporated herein by reference. For example, polyethylene glycol can be covalently attached by amino acid residues via reactive groups such as free amino or carboxyl groups. A reactive group is a group to which an activated polyethylene glycol molecule can be attached. An amino acid residue having a free amino group may include a lysine residue and an N-terminal amino acid residue, and an amino acid residue having a free carboxyl group may include an aspartic acid residue, a glutamic acid residue and a C-terminal amino acid residue. Get included. Sulfhydryl groups can also be used as a reactive group for attaching polyethylene glycol molecules. Preferred for therapeutic purposes are attachments at amino groups such as attachments at the N-terminus or lysine groups. It may be particularly desirable to chemically modify the protein at the N-terminus. When polyethylene glycol is used to describe this composition, various polyethylene glycol molecules (depending on molecular weight, branching, etc.), the ratio of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mixture, and the type of PEGylation reaction used , As well as methods for obtaining the N-terminal PEGylated protein of choice. A method for obtaining N-terminal PEGylation preparations (ie, separating this moiety from other monoPEGylated moieties as needed) is described by N-terminal PE from a population of PEGylated protein molecules.
It may also be a purification of the G compound. Selected proteins chemically modified in the N-terminal modification utilize reductive alkylation, which takes advantage of the specific reactivity of different types of primary amino groups (lysine vs. N-terminal) available for derivatization in specific proteins. Can be achieved. Under suitable reaction conditions, depending on the carbonyl group containing polymer
Substantially selective derivatization of the protein at the N-terminus is achieved.

<Multimerization> The polypeptide of the present invention is a monomer or multimer (ie, dimer, trimer,
Tetramers and higher multimers). Accordingly, the invention relates to monomers and multimers of the polypeptides of the invention, methods for their preparation, and compositions containing them.
In particular embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In other embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers. Multimers encompassed by the present invention can be homomers or heteromers. As used herein, "homomer" refers to the amino acid sequences of SEQ ID NOs: 242-482 or the amino acid sequences encoded by the clone inserts of the deposited clone pool (for these polypeptides as described herein). Corresponding fragments, variants, splice variants, and fusion proteins). These homomers may include polypeptides having the same or different amino acid sequences. In certain embodiments, homomers of the invention are multimers that only include polypeptides that have the same amino acid sequence. In another particular embodiment, the homomers of the invention are multimers that include only polypeptides having different amino acid sequences. In certain embodiments, homomers of the invention are homodimers (eg, including polypeptides having the same or different amino acid sequences) or homotrimers (eg, polypeptides having the same and / or different amino acid sequences). Is included). In other embodiments, the homomeric multimers of the invention are at least homodimers, at least homotrimers, or at least homotetramers. As used herein, "heteromer" means a multimer comprising one or more heterologous polypeptides (ie, polypeptides of different proteins) in addition to the polypeptide of the present invention. In certain embodiments, the multimers of the invention are heterodimers, heterotrimers, or heterotetramers. In other embodiments, the heteromeric multimers of the invention are at least heterodimers, at least heterotrimers, or at least heterotetramers. The multimers of the invention can be the result of hydrophobic, hydrophilic, ionic and / or covalent associations, and / or indirect association, eg by liposome formation. Thus, in one embodiment, multimers of the invention, eg, homodimers or homotrimers, are formed when the polypeptides of the invention contact each other in solution. In another embodiment, a heteromultimer of the invention, eg, a heterotrimer or heterotetramer, comprises an antibody (a fusion of the invention of the invention) against which the polypeptide of the invention is in solution. Formed upon contact with (including antibodies to heterologous polypeptide sequences in proteins). In other embodiments, the multimers of the invention are formed by covalent association with and / or between the polypeptides of the invention. Such covalent association involves the inclusion of one or more amino acid residues contained in the polypeptide sequence (eg, listed in the sequence listing or contained in the polypeptide encoded by the deposited clone). Including. In some cases, the covalent association is a bridge between cysteine residues located within the polypeptide sequence that interacts with the native (ie, naturally occurring) polypeptide. In other cases, covalent association is the result of chemical or recombinant engineering treatments. Alternatively, such a covalent association may involve one or more amino acid residues contained in the heterologous polypeptide sequence of the fusion protein of the invention.

In one example, covalent association occurs between heterologous sequences contained in a fusion protein of the invention (see, eg, US Pat. No. 5,478,925, the disclosure of which is incorporated herein by reference in its entirety. In a particular example, the covalent association occurs between heterologous sequences contained in the Fc fusion proteins of the invention (as described herein). In other specific examples, covalent association of a fusion protein of the invention is described, for example, in oseteoprotegerin (see, eg, International Patent Application Publication WO 98/49305, the disclosure of which is incorporated herein by reference in its entirety). Between the heterologous polypeptide sequences from other proteins capable of forming covalently associated multimers (e.g. In other embodiments, two or more polypeptides of the invention are linked by a peptide linker. Examples include the peptide linkers described in US Pat. No. 5,073,627 (incorporated herein by reference). Proteins comprising multiple polypeptides of the invention separated by a peptide linker can be produced using conventional recombinant DNA technology. Other methods of preparing the multimeric polypeptides of the present invention extend to the use of the polypeptides of the present invention fused to leucine zipper or isrosin zipper polypeptide sequences. Leucine and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are present. The leucine zipper was first identified among several DNA binding proteins and has since been found in a variety of different proteins (Landschulz et al., 1988). Known leucine zippers include naturally occurring peptides and their dimerized or trimerized derivatives. Examples of leucine zipper domains suitable for producing the soluble multimeric proteins of the invention are those described in International Patent Application Publication WO 94/10308,
Incorporated herein by reference. A recombinant fusion protein comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimers in solution is expressed in a suitable host cell and the resulting soluble multimer. The fusion protein is recovered from the culture supernatant using techniques known in the art. The trimeric polypeptides of the present invention may provide the advantage of enhancing biological activity. Preferred leucine zipper moieties and isoleucine moieties are those that selectively form trimers. One example is Hoppe et al. (1994) and US patent application Ser. No. 08 / 446,922.
Is a leucine zipper derived from lung surfactant protein D (SPD), the disclosure of which is incorporated herein by reference in its entirety. Other peptides derived from naturally occurring trimeric proteins can be used to prepare the trimeric polypeptides of the invention. In another example, a protein of the invention is associated by interaction between Flag® polypeptide sequences contained in a fusion protein of the invention that includes a Flag® polypeptide sequence. In a further embodiment, an associated protein of the invention is associated by interaction between a heterologous polypeptide sequence comprising a Flag® fusion protein of the invention and an anti-Flag® antibody.

The multimers of the invention can be produced using chemical techniques known in the art. For example, the polypeptides desired to be included in the multimers of the present invention can be chemically crosslinked using linker molecules and linker molecule length optimization techniques known in the art (see US Pat. No. 5,478,925). The entire publication is incorporated by reference into the present specification). In addition, the multimers of the invention form one or more intermolecular bridges between cysteine residues located within the polypeptide sequence that it is desired to include in the multimer using techniques known in the art. (Eg, US Pat. No. 5,478, which is incorporated by reference herein in its entirety).
See No. 925). Further, the polypeptide of the present invention may be the C-terminus of the polypeptide or
Generally modified by adding cysteine or biotin to the N-terminus, 1
Alternatively, techniques known in the art may be applied to produce multimers comprising more than one of these modified polypeptides (e.g., incorporated herein by reference in its entirety). See US Pat. No. 5,478,925). In addition, 30 techniques known in the art can be applied to produce liposomes containing polypeptide components that it is desirable to include in the multimers of the invention (see, eg, the entire text herein). (See US Pat. No. 5,478,925). Alternatively, the multimers of the present invention can be produced using genetic engineering techniques known in the art. In one embodiment, the polypeptides included in the multimers of the invention are recombinantly produced using the fusion protein technologies described herein or known in the art. (See, eg, US Pat. No. 5,478,925, the entire text of which is incorporated herein by reference). In a particular embodiment, the polynucleotide encoding the homodimer of the invention has a polynucleotide sequence encoding a polypeptide of the invention linked to a sequence encoding a linker polypeptide, which in turn further comprises Produced by binding to a synthetic polynucleotide (lacking a leader sequence) that encodes the translation product of a polypeptide that is oriented in the opposite orientation from the C-terminus to the N-terminus (see, eg, its entire text herein). (See US Pat. No. 5,478,925, incorporated by reference). In other embodiments,
Recombinant techniques described herein or known in the art include transmembrane domains (or hydrophobic or signal peptides) of the invention that can be incorporated into liposomes by membrane reconstitution methods. It has application to produce recombinant polypeptides (see, eg, US Pat. No. 5,478,925, incorporated herein by reference in its entirety).

Mutant Polypeptides Protein engineering can be utilized to improve or alter the characteristics of the GENSET polypeptides of the present invention. Utilizing recombinant DNA technology known to those skilled in the art,
New muteins, including single or multiple amino acid substitutions, deletions, additions, or fusion proteins, or muteins can be created. Such modified polypeptides can exhibit, for example, increased / reduced biological activity, or increased / reduced stability. Moreover, they can be purified in high yields and, at least under certain purification and storage conditions, exhibit greater solubility than the corresponding native polypeptides.
Furthermore, the polypeptides of the present invention can be produced as multimers, including dimers, trimers, and tetramers. Multimerization can be facilitated by a linker or recombination via a heterologous polypeptide such as the Fc region. N- and C-Terminal Deletions It is known in the art that one or more amino acids can be deleted from the N-terminus or C-terminus without substantial loss of biological function. For example, Ron et al. (1993)
Is a modified KGF, even in the absence of the 3, 8 or 27 N-terminal amino acid residues.
It was reported that the protein has heparin binding activity. Accordingly, the invention provides the polypeptides of SEQ ID NOS: 242-482, or the deposited clone pools in which one or more residues have been deleted from the amino terminus of the polypeptide encoded by the clone insert. Similarly, numerous examples of biofunctional C-terminal deletion mutations are known. For example, interferon gamma is
Deletion of 810 amino acid residues from the C-terminus of the protein results in up to 10-fold higher activity (see, eg, Dobeli, et al. 1988. The disclosure of which is incorporated herein by reference in its entirety. ). Accordingly, the invention provides a polypeptide represented by SEQ ID NOs: 242-482, or a polypeptide that deletes one or more residues from the carboxy terminus of the polypeptide encoded by the clone insert into the deposited clone pool. . The present invention also provides polypeptides that have one or more amino acid deletions from both the amino and carboxyl termini, as described below. <Other Mutations> The present invention includes other mutants in addition to the N- and C-terminal deletion forms of the above protein. It will also be appreciated by those skilled in the art that certain amino acid sequences of the GENSET polypeptides of the present invention can be mutated without substantially affecting the structure or function of the protein. It should be understood that the key regions that determine activity are on proteins where such differences in sequence are designed. Accordingly, the invention further includes mutations in the GENSET polypeptide that exhibit substantially GENSET polypeptide activity. Such mutants may be deletions, insertions, inversions, repeats, selected according to general rules known in the art, such that they have little effect on activity.
And includes substitutions. For example, guidelines have been given on how to create phenotypically silent (silent) amino acid substitutions.

There are two main ways to study the permissiveness of amino acid sequences for changes (see Bowie et al. 1994), the disclosure of which is incorporated herein by reference in its entirety. The first method is based on an evolutionary process in which mutations are accepted or rejected by natural selection. The second method utilizes genetic engineering to derive amino acid changes at specific positions of the cloned gene, as well as selections or screens to identify sequences that maintain functionality. These studies have shown that the protein is surprisingly permissive for amino acid substitutions. These studies show that amino acid changes are likely to be tolerated at certain positions in the protein. For example, surface side chain features are generally poorly conserved, whereas most buried amino acid residues require non-polar side chains. Other such phenotypic silent substitutions are described by Bowie et al.
(Supra) and is incorporated herein by reference. The commonly observed conservative substitutions are the aliphatic amino acids Ala, Val, Leu and Phe.
Conversion of one to another between; the interchange of hydroxyl groups of Ser and Thr, As
Exchange of p and Glu acidic residues, substitution between Asn and Gln amide residues, Lys and A
Exchange of basic residues of rg and substitution between aromatic residues of Phe and Try. Thus, a fragment, derivative, composition, or homologue of a polypeptide of the invention may include, for example: (i) one or more amino acid residues that are conservative or non-conservative Residues) and whether such substituted amino acid residues are amino acid residues encoded by the genetic code: or (ii) one or more amino acid residues Containing substituents: or (iii) a GENSET polypeptide fused to another compound, such as a compound that increases the half-life of the polypeptide (eg, polyethylene glycol): or (iv) other amino acids Is the IgG Fc fusion region peptide or leader or secretory sequence or sequence or protein used to purify the polypeptide in the form described above. Such as Park protein sequence, which is fused to a polypeptide of the above forms. Such fragments, derivatives and compositions are considered to be within the understanding of those of ordinary skill in the art in view of the teachings herein. Accordingly, the GENSET polypeptides of the present invention may include one or more amino acid substitutions, deletions, or additions due to natural mutations or engineered by humans. As explained, the changes are preferably of non-critical nature, such as conservative amino acid substitutions that do not substantially affect the folding or activity of the protein.
The following groups of amino acids generally show equivalent changes: (1) Ala, Pro, Gly, Glu,
Asp, Gln, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, Ile,
Leu, Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Tyr, Trp, His
.

A particular embodiment for a modified GENSET peptide molecule of interest according to the invention is that the peptide molecule that is resistant to proteolysis, the -CONH-peptide bond is modified,
And, but not limited to, (CH2NH) reducing bond, (NHCO) reverse-inverting bond, (CH2-O
) Methylene-oxy bond, (CH2-S) thiomethylene bond, (CH2CH2) carba bond, (CO
-CH2) including a peptide substituted by a cetomethylene bond, a (CHOH-CH2) hydroxyethylene bond), a (NN) bond, an E-arsene bond or a -CH = CH- bond. The invention also includes a human GENSET polypeptide or a fragment or variant thereof in which at least one peptide bond has been modified as described above. The amino acids of the GENSET protein of the invention that are essential for function are site-directed or alanine scanning mutagenesis (eg, Cunningh).
(see am et al. 1989)), the disclosures of which are incorporated herein by reference in their entirety. The latter method induces a single alanine mutation at every residue in the molecule. The resulting mutant molecule is then tested for biological activity using an assay suitable for measuring the function of that particular protein. The replacement of charged amino acids with other charged or neutral amino acids is of particular interest so that proteins with highly desirable improved characteristics, such as less aggregation, can be produced. Aggregation can be problematic in the preparation of pharmaceutical formulations because aggregates can be immunogenic rather than merely reducing activity (eg, Pinckard et al., 1967; Robbins et al., 1987; and Cleland et al., 1993).
See). A further embodiment of the invention is at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, not more than 40 conservative amino acid substitutions, not more than 30 conservative amino acid substitutions, and more than 20 A polypeptide comprising an amino acid sequence of a GENSET polypeptide having an amino acid sequence comprising a conservative amino acid substitution which is not. Also, at least one, but 10, 9, 8, 7, 6, 5, 4, 3
Provide a polypeptide comprising an amino acid sequence of a GENSET polypeptide having no, two, or more than one conservative amino acid substitution. Polypeptide Fragments <Structure Definition> The present invention further provides polypeptides of SEQ ID NOS: 242-482, SEQ ID NOS: 242-272 and
274-384, fragments of the amino acid sequences described herein, such as the mature polypeptides, full length or mature polypeptides encoded by the clone inserts of the deposited clone pool. More specifically, the invention provides a polypeptide selected from the group consisting of the sequences of SEQ ID NOs: 242-482, SEQ ID NOs: 242-272.
And the mature proteins contained in 274-384, and full-length or mature polypeptides encoded by clone inserts in the deposited clone pool, and other polypeptides of the invention, at least 6, preferably at least 8-10,
More preferably 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150
, Purified, isolated, and recombinant polypeptides containing 200, 250, 300, 350, 400, 450 or 500 contiguous amino acids.

In addition to the above polypeptide fragments, a further preferred subgenus of polypeptides comprises at least 6 amino acids, where "at least 6" comprises 6 and the polypeptide sequences listed below in the sequence listing. It is defined as an integer in the range of integers that represents the C-terminal amino acid of a polypeptide of the invention. Also included are polypeptide fragment species that are at least 6 amino acids in length, further specified for N-terminal and C-terminal positions, as described above. However, included as individual species in the present invention are all polypeptide fragments that are at least 6 amino acids in length, as explained above, and may be specified by N-terminal and C-terminal positions in particular. That is, the present invention includes all combinations of N-terminal and C-terminal positions that can be occupied by fragments that are above the sequence listing or a given amino acid sequence of the invention and that are at least 6 contiguous amino acid residues in length. . The present invention also provides for exclusion of fragment species specified by N-terminal and C-terminal positions, or fragment subgenus specified by amino acid residue size, as described above. Any number of fragments identified by N-terminal and C-terminal positions or amino acid residue size as described above can be excluded as a separate species. The above polypeptide fragments of the present invention are not listed individually individually for the purpose of being observed contiguously using the above description and thus not unnecessarily lengthening the specification. Further, a biologically active polypeptide is a preferred embodiment of the present invention, for example, immunoassay, epitope mapping, epitope tagging,
To be useful as a vaccine or as a molecular weight marker, the above fragments need not have GENSET bioactivity. The fragments can also be used to raise antibodies to specific portions of the polypeptide. These antibodies are then used in the art to discriminate between human and non-human cells and tissues, or to determine if cells or tissues in a biological sample are the same type expressing a polypeptide of the invention. Can be used in the immunoassay method known in. It is noted that the above species of polypeptide fragments of the invention can alternatively be described by formulas "ab"; where "a" is the N-terminal maximum amino acid position of the polynucleotide and "b" is the C-terminal maximum amino acid position. Equal to; and "a" is
1 and an integer in the number of amino acids of the polypeptide sequence of the invention minus 6 and "b" equals an integer in the range of 7 and the number of amino acids of the polypeptide sequence of the invention; and "A" is at least 6 less than "b".

The present invention also provides for the exclusion of species of the polypeptide fragments of the present invention, identified by a subgenus of polypeptides identified by 5'and 3'positions or amino acid size as described above. Supply. As explained above, all fragment numbers specified by 5'and 3'positions or amino acid size can be excluded. Particularly excluded from the present invention are polypeptide fragments encoded by the selectively excluded polynucleotide fragments set forth in Table IV and Tables Va and Vb. Table IV, and Tables Va and Vb, in addition to those described elsewhere in this specification, separately show the exclusion of polypeptides and are therefore not intended to be limiting. Functional Definitions Preferred polypeptide fragments of the invention are represented by signal peptides, preferably a signal peptide selected from the group consisting of SEQ ID NOs: 242-272 and 274-384, SEQ ID NOS: 1-31 and 33-143. An isolated, purified or recombinant polypeptide comprising, consisting of, or consisting essentially of, the encoded signal peptide as well as the signal peptide encoded by the clone insert of the deposited clone pool. Such polypeptide fragments are useful for designing secretory vectors as described elsewhere in this application. Other preferred polypeptide fragments of the invention are encoded by mature proteins, preferably mature proteins selected from the group consisting of SEQ ID NOs: 242-272 and 274-384, SEQ ID NOs: 1-31 and 33-143. An isolated, purified or recombinant polypeptide comprising, consisting of, or consisting essentially of, a mature protein as well as a mature protein encoded by a clone insert of the deposited clone pool. [Domain] A preferred polynucleotide fragment of the present invention is a polypeptide domain of the present invention. Such domains are ultimately leucine zippers, helix-
Post-translational modification sites such as turn-helix motifs, glycosylation sites, ubiquitin binding sites, alpha-helices and beta-sheets, signal sequences encoding signal peptides that direct the secretion of encoded proteins, homeoboxes, acidic stretches, enzymatic activity Includes linear or structural motifs and markings including, but not limited to, sites involved in transcriptional regulation such as sites, substrate binding sites, and enzyme cleavage sites. Such domains may exhibit specific biological activities such as DNA or RNA binding, protein secretion, transcriptional regulation, enzymatic activity, substrate binding activity and the like.

Domains generally have a size comprised between 3 and 2000 amino acids. In a preferred embodiment, the domain comprises a number of amino acids that is an integer in the range 6-500. Domains can be synthesized using any of the methods known to those of skill in the art, including those disclosed herein, particularly those disclosed in the section entitled "Preparation of Polypeptides of the Invention". Methods to determine the amino acids that make up a domain with a particular biological activity include mutagenesis studies and detection methods to determine the biological activity to be tested. Alternatively, the polypeptides of the invention can be scanned for motifs, domains and / or indicia in a database using any of the computer methods known to those of skill in the art. Searchable databases are Prosite (Hofmann et al., 1999;
Bucher and Bairoch 1994), Pfam (Sonnhammer et al., 1997; Henikoff et al., 2000.
Bateman et al., 2000), Blocks (Henikoff et al., 2000), Print (Attwood et al., 1996).
, Prodom (Sonnhammer and Kahn, 1994; Corpet et al 2000), Sbase (Pongor et al, 1993; Murvai et al, 2000), Smart (Schultz et al, 1998), Dali / FSSP (Holm and Sander, 1996, 1997 and 1999), HSSP (Sander and Schneider 1991), CAT
H (Orengo et al., 1997; Pearl et al., 2000), SCOP (Murzin et al., 1995; Lo Conte et al., 2
000), COG (Tatusov et al., 1997 and 2000), specific species databases and their derivatives (Nevill-Manning et al., 1998; Yona et al., 1999; Attwood et al., 2000), the entire disclosure of each of which is hereby claimed. It is incorporated by reference in the description. For a survey of available databases, see Nucleic Acid Research (2000), vol 28, first edition. The entire disclosure of which is incorporated herein by reference. The polypeptides of SEQ ID NOs: 242-482 were screened for the presence of known structural or functional motifs or the presence of small amino acid sequences that are well conserved between protein species. Search HMMER-2.1.1 (for information, go to S
onnhammer et Durbin, HYPERLINK "http: /www.sanger.ac.uk/Pfam/" http: / www
.sanger.ac.uk / Pfam /), in the Pfam5.5 database, in the emotif (for information Nevill-Manning et al., PNAS, 95, 5865-5871, (1998), HYPERLINK "ht
tp: //motif.stanford/edu/EMOTIF)) "http: //motif.stanford/edu
/ EMOTIF), Blocks version 12.0, Prints version 26.0, P
Blocks Plus database including fam version 5.3, Prodom version 99.1, and Domo version 2.0, and bla (Tatusov, RL & Koonin, E.
V. CABIOS 10, No. 4) and pfscan (http://www.isrec.isb-sib.ch/cgi-bin/m
an.cgi-section = 1 & topic = pfscan) with Prosite 16.0 database. Table VI describes some of these prediction domains. For those polypeptides referenced by their SEQ ID NOs (columns entitled "SEQ ID NO"), the database of domains shown in the column entitled "Database" and preferred fragment positions within these sequences (titled "domain positions") Table VI shows the designated domains (columns labeled "Specified Domains") according to columns. Each fragment is designated by ab, where a and b are the start and end positions, respectively, of a given preferred fragment on the full length polypeptide. Preferred fragments are separated from each other by commas. As used herein, "domains described in Table VI" means all domains listed in Table VI for a given GENSET protein referenced by SEQ ID NO: in the first column. It should be noted that in Table VI, the first encountered methionine is designated as amino acid number 1, ie the leader sequence is not negatively numbered. In the appendix sequence listing, the first amino acid of the mature protein by signal peptide cleavage is designated as amino acid number 1 according to the management rules of the sequence listing,
The first amino acid of the signal peptide is designated by an appropriate negative number.

Finally, the preferred polynucleotide fragments of the present invention are SEQ ID NOS: 242-482.
Is a polypeptide domain of. Accordingly, the present invention is a sequence selected from the group consisting of the sequences of SEQ ID NOs: 242-482, preferably at least 6, preferably 8-10, more preferably 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 125, 150, 175
, 200, 225, 250, 275, 300, 350, 400, 450 or 500 amino acid contiguous spans, consisting of, predominantly or including, contiguous spans of these lengths with the length of the selected sequence. An isolated, purified or recombinant polypeptide of matching size, said contiguous spans comprising at least 1, 2, 3, 5, or 10 amino acid positions of a domain described in Table VI of said selected sequence. Includes. The present invention also includes at least a sequence selected from the group consisting of the sequences of SEQ ID NOs: 242-482.
, Preferably at least 8-10, more preferably 12, 15, 20, 25, 30, 35, 4
0, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 4
Consisting of, predominantly, or including contiguous spans of 00, 450 or 500 amino acids, contiguous spans of these lengths sized to match the length of the selected sequence, said contiguous spans comprising: Included are isolated, purified or recombinant polypeptides that are the domains described in Table VI of the selection sequences. The present invention also includes an isolated, purified or recombinant polypeptide comprising, consisting of, or consisting essentially of, the domains set forth in Table VI of sequences selected from the group consisting of the sequences of SEQ ID NOs: 242-482. Include. Polypeptides of the invention not specifically described in this table are not considered to be domain-attached. This is because it may not be perceived as likely by the particular algorithm used, or may not be included in the particular database searched. In fact, all fragments of the polypeptides of the invention that are at least 6 amino acid residues in length are included in the invention as domains. Amino acid residues containing other domains can be determined by searching databases other than those currently cited to establish Table VI. A domain of the invention comprises 6 to 200 amino acids (ie, 6 to 200 amino acids) that are polypeptides of the invention.
(Including an integer in the range of) is preferably included. In addition, the present invention provides 6 and the full length G of the sequence listing.
It also includes domain fragments in the range of integers that are ENSET arrays. All combinations of sequences in the integer range consisting of 6 and the full length sequence of the GENSET polypeptide are included.
A domain fragment can be a number of contiguous amino acid residues (as a subgenus) or a specific N-terminus and C, as described above for polypeptide fragments of the invention.
It can be identified by either end position (as a species). The number of domain fragments of the invention can also be excluded in a similar manner.

Epitope and Antibody Fusions: A preferred embodiment of the invention relates to epitope containing polypeptides and epitope containing polypeptide fragments. These epitopes can be "antigenic epitopes" or both "antigenic epitopes" and "immunogenic epitopes". An "immunogenic epitope" is defined as the part of a protein that elicits an antibody response in vivo when the polypeptide is the immunogen. On the other hand, the polypeptide region that an antibody binds is defined as an "antigenic determinant" or "antigenic epitope." The number of immunogenic epitopes of a protein is generally lower than the number of antigenic epitopes (see, eg, Geysen et al., 1984, the disclosure of which is incorporated herein by reference in its entirety). It may not be immunogenic, but it is specifically noted that antibodies are still useful because they can generate both immunogenic and antigenic epitopes. An epitope can include three amino acids, which are in the least spatial conformation, which is unique to the epitope. Generally, an epitope consists of at least 6 such amino acids and often at least 8-10 such amino acids. In a preferred embodiment, the antigenic epitope comprises a number of amino acids that is an integer in the range 3-50. Fragments that function as epitopes can also be produced by conventional means (see, eg, Houghten, 1985) and are further described in US Pat.
4,631,21, the disclosure of which is incorporated by reference in its entirety. Methods for determining the amino acids that make up the epitope are described in X
Line crystallography, two-dimensional nuclear magnetic resonance, and epitope mapping, examples of which are:
Pepscan method described by Geysen et al. (1984); International Patent Application Publication WO84 / 03
No. 564; and International Patent Application Publication No. WO84 / 03506, the disclosures of which are incorporated by reference in their entireties. Another example is the algorithm of Jameson and Wolf (1988), which is incorporated by reference in its entirety.
This Jameson-Wolf antigenic analysis, for example, uses default parameters (version
4.0 Windows, DNASTAR, Inc., 1228 South Park Street Madison, WI.)
Can be carried out using the computer program PROTEAN. The antigenic epitopes predicted by the Jameson-Wolf algorithm for the polypeptides of SEQ ID NOS: 242-482 are shown in Table VII. For each GENSET polypeptide referred to by a SEQ ID NO: in the column entitled "SEQ ID NO:", a listing of antigenic epitopes is shown in the column entitled "Epitope", each epitope separated by a comma. Each fragment is designated by ab, where a and b are the start and end positions, respectively, of a given preferred fragment. Table VII
It should be noted that in, the first encountered methionine is designated as amino acid number 1, ie the leader sequence is not negatively numbered. In the appendix sequence listing, the first amino acid of the mature protein by signal peptide cleavage is designated as amino acid number 1, and the first amino acid of the signal peptide is designated by an appropriate negative number according to the management rules of the sequence listing. As used herein, see Table VII.
The "epitope described in" refers to the GENSET referred to by the SEQ ID NO in the first column.
For polypeptide, we mean all preferred polynucleotide fragments described in the second column of Table VII. The immunogenic epitopes listed in Table VII are:
It has been shown that specific algorithms only describe the amino acid residues that comprise the epitope that are predicted to be the most immunogenic. Polypeptides of the invention not specifically described as immunogenic are not considered non-antigenic. This is because they are antigenic in vivo but were not recognized as antigenic only by the particular algorithm used. Alternatively, polypeptides are often antigenic in vitro using methods such as phage display. Therefore,
Listed in Table VII are amino acid residues containing only preferred epitopes,
Not a complete list. Indeed, all fragments of the polypeptides of the present invention that are at least 6 amino acid residues in length are included in the present invention as being useful as antigenic epitopes. Amino acid residues containing other immunogenic epitopes are Ja
It can be determined by in vivo testing for antigenic response using an algorithm similar to meson-Wolf analysis or the methods described herein or methods known in the art.

Therefore, the present invention is a sequence selected from the group consisting of the sequences of SEQ ID NOs: 242-482, at least 6, preferably 8-10, more preferably 12, 15, 20, 25, 30,
35, 40, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 3
Consisting of, consisting predominantly of, or including contiguous spans of 50, 400, 450 or 500 amino acids, the contiguous spans of these lengths having a size corresponding to the length of the selected sequence, said contiguous spans Include isolated, purified or recombinant polypeptides comprising at least 1, 2, 3, 5, or 10 amino acid positions of the epitopes set forth in Table VII of the selected sequences above. The present invention is also a sequence selected from the group consisting of the sequences of SEQ ID NOs: 242-482, preferably at least 6, preferably at least 8
10, more preferably 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 12
Consisting of, consisting predominantly of, or including contiguous spans of 5, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450 or 500 amino acids, contiguous spans of these lengths being Includes isolated, purified or recombinant polypeptides that are sized to match the length of the selected sequence and said flanking spans are the epitopes described in Table VII of the selected sequence. The invention also includes an isolated, purified or recombinant polypeptide comprising, consisting of, or consisting essentially of, the epitopes set forth in Table VII of sequences selected from the group consisting of the sequences of SEQ ID NOs: 242-482. To do. Epitope-containing fragments of the invention are polypeptides of the invention 6-50
Amino acids (ie, including integers in the range 6-50) are preferably included. The present invention also includes antigenic fragments in the range of 6 and the full length GENSET sequence in the sequence listing. All combinations of sequences in the integer range consisting of 6 and the full length sequence of the GENSET polypeptide are included. Epitope-containing fragments can be identified by either the number of contiguous amino acid residues (as a subgenus) or specific N- and C-terminal positions (as a species), as described above for polypeptide fragments of the invention. . The number of epitope-containing fragments of the invention can also be excluded in a similar manner. Antigenic epitopes are useful for producing antibodies, including monoclonal antibodies that specifically bind to the epitope (Wilson et al., 1984; and Sutcliffe et al.,
1983), the entire disclosure of which is incorporated herein by reference. The antibodies are then used in various techniques, such as diagnostic and tissue / cell identification techniques, and purification methods, such as immunoaffinity chromatography, as described herein.

An antibody or other compound that specifically binds a polypeptide or polynucleotide of the invention can also be said to "selectively recognize" a polypeptide or polynucleotide. Similarly, immunogenic epitopes can be used to induce antibodies by methods known in the art (Sutcliffe et al., Supra; Wilson et al., Supra; Chow et al.
(1985) and Bittle et al., (1985). The entire disclosures of which are incorporated herein by reference). Preferred immunogenic epitopes are natural GENS
Contains ET protein. Immunogenic epitopes (such as rabbit or mouse)
It can occur with a carrier protein such as albumin for animal systems, or if the length is sufficiently long (at least about 25 amino acids), without a carrier. However, if at most, an immunogenic epitope containing only 8-10 amino acids is sufficient to produce an antibody capable of binding at least a linear epitope present in the denatured polypeptide (eg, Western blotting). It is shown. Epitope-containing polypeptides of the invention include, but are not limited to, in vivo immunization, in vitro immunization, and phage display (eg, Sutcliffe et al., Supra; Wilson et al., Supra; and Bittle et al., Supra). It is used to induce antibodies by methods known in the art including. When using the in vivo immunization method, animals are immunized with the free peptide but the anti-peptide antibody titer is increased by conjugating the peptide to a polymeric carrier such as keyhole limpet hemocyanin (KLH) or tetanus toxin. You may boost. For example, a peptide containing a cysteine residue is -maleimidobenzoyl-N-hydroxysuccinimide ester (MB
While linkers such as S) can be used to conjugate to the carrier, other peptides can be conjugated to the carrier using more common binders such as glutaraldehyde. Animals such as rabbits, rats and mice can be treated with free or carrier-conjugated peptide by, for example, intraperitoneal and / or intradermal injection of an emulsion (emulsion) containing about 100 (g of peptide or carrier protein and Freund's adjuvant. Immunization, eg, to generate an effective titer of an anti-peptide antibody that can be detected by an ELISA assay using free peptide absorbed on a solid surface, eg, at intervals of about 2 weeks Booster injections may be required.Anti-peptide antibody titers in serum from immunized animals may be determined by methods known in the art, such as absorption to peptides on a solid support and elution of selected antibodies. It can be increased by selection of anti-peptide antibody.

Those of skill in the art will understand that the polypeptides of the present invention that include immunogenic or antigenic epitopes can be fused to heterologous polypeptide sequences and will consider as described above. For example, the polypeptides of the present invention are immunoglobulins (IgA, IgE, Ig
G, IgM), or a portion thereof (CH1, CH2, CH3, a combination including both whole domains and portions thereof) constant domain may result in a chimeric polypeptide. These fusion proteins facilitate purification and exhibit increased in vivo half-life. This has been shown, for example, for chimeric proteins consisting of the first two domains of human CD4-polypeptide and various domains of the constant region of the heavy or light chains of mammalian immunoglobulins (eg EPA 0,394,827; and Tr
aunecker et al., 1988), the disclosures and disclosures of which are incorporated herein by reference in their entirety. Fusion proteins with a disulfide-bonded dimeric structure due to the IgG portion are also more efficient in binding and neutralizing other molecules as compared to the monomeric polypeptide or fragments thereof alone (eg, Fountoul.
akis et al., 1995), the entire disclosure of which is incorporated herein by reference. Nucleic acids encoding the above epitopes can also be recombined with the gene of interest as an epitope tag to facilitate detection and purification of the expressed polypeptide. Other fusion proteins of the invention can be produced by gene shuffling, motif shuffling, exon shuffling, or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to modulate the activity of the polypeptides of the invention, thereby effectively producing polypeptide agonists and antagonists. For example, US Pat.
5,793; 5,811,238; 5,834,252; 5,837,458 and Patten et al. (1997).
Harayama, (1998); Hansson, et al., (1999); and Lorenzo and Blasco, (
1998) (each of these documents is incorporated herein by reference). In one embodiment, the one or more components, motifs, sections, parts, domains, fragments, etc. encoding the polynucleotides of the invention, or the polypeptides encoded thereby, are of one or more heterologous molecules. It can be recombined with one or more components, motifs, sections, parts, domains, fragments.

The present invention further includes any combination of the polypeptide fragments listed in this section. Antibodies: <Definition> The invention further provides antibodies and T cell antigen receptors that specifically bind to a polypeptide, and more specifically to an epitope of a polypeptide of the invention.
Regarding (TCR). Antibodies of the invention include IgG (including IgG1, IgG2, IgG3, and IgG4) IgA (including IgA1 and IgA2) IgD, IgE, or IgM, and IgY. An "antibody" (Ab) comprises at least one binding domain, which is formed by the folding of the variable domain of the antibody molecule and which has an internal surface shape and charge distribution complementary to the characteristics of the antigenic determinants of the antigen. Forming a three-dimensional combined space equipped with,
By a polypeptide or a group of polypeptides, which allows an immunological reaction with an antigen. As used herein, "antibody" means single chain whole antibody,
And antigen-binding fragments thereof are meant to include whole antibodies. In preferred embodiments, the antibody includes, but is not limited to, Fab, Fab ′ F (ab) 2 and
F (ab ') 2, Fd, single chain Fvs (scFv), single chain antibody, disulfide bond Fvs (sdFv
) And a fragment comprising a V _L or V _H domain. The antibody may be of any animal origin including birds and mammals. Preferably, the antibody is from human, mouse, rabbit, goat, guinea pig, camel, horse, or chicken. Antigen-binding antibody fragments, including single chain antibodies, are variable region (s)
Alternatively, it may be included alone or in combination with all or part of the hinge region, CH1, CH2, and CH3 domains. The present invention also includes combinations of variable region (s) and hinge regions, CH1, CH2, and CH3 domains. The invention further includes chimeric, humanized, and human monoclonal and polyclonal antibodies that specifically bind the polypeptides of the invention. The present invention further includes antibodies that are anti-idiotype (anti-specific) to the antibodies of the present invention. Antibodies of the invention may be monospecific, bispecific, and trispecific, or of higher order multispecificity. A multispecific antibody is specific for different epitopes of a polypeptide of the invention or for both a polypeptide of the invention as well as a heterologous polypeptide or a heterologous composition such as a solid support material. . For example, International Patent Application Publication WO 93/17715; WO 92/08802; WO 91/00360;
WO 92/05793; Tutt et al. (1991); U.S. Patents 5,573,920, 4,474,893, 5,601,819.
, 4,714,681, 4,925,648; Kostelny et al. (1992). The entire publication is incorporated herein by reference.

[0078]   Antibodies of the present invention are those that are identified by or are specifically bound by the antibody.
Description of the epitope or epitope-containing portion of the polypeptide of the invention or
Can be specified. An antibody is a nucleic acid of the invention, or a fragment thereof, especially a secretory protein.
In the case of proteins, the complete protein encoded by the mature protein or signal peptide.
It can bind specifically to all proteins. Therefore, the epitope (s)
Is the epitope-containing polypeptide moiety (s), eg, N-terminal and C
Terminal position, size of adjacent amino acid residues, or otherwise as described herein (
Sequence listings)) can be identified as described herein. Epito of the present invention
Antibody that specifically binds to either the
Can be eliminated. Therefore, the present invention specifically binds to a particular polypeptide of the present invention.
Includes matching antibodies, allowing elimination of the like.   Therefore, another embodiment of the present invention is the sequence of SEQ ID NOs: 242-482 and the deposited clone
A polype containing a sequence selected from the group consisting of sequences of pooled clone inserts.
A purified or isolated antibody capable of specifically binding to a peptide. This
In one aspect of this embodiment, the antibody is SEQ ID NOS: 242-482 and the deposited clone clone.
Is a sequence selected from the group consisting of
Together with 6 contiguous amino acids, preferably at least 8-10 contiguous amino acids, and
Preferably comprises 12, 15, 20, 25, 30, 40, 50 or 100 contiguous amino acids
, Is capable of binding to an epitope-containing polypeptide.   The antibodies of the present invention may also be described or specified with respect to their cross-reactivity. The present invention
To specifically bind other analogs, orthologs, or homologs of
No antibodies included. 95% or less and 90% or less identity with the polypeptide of the present invention
Lower, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less 55% or less,
And up to 50% (methods known in the art and described herein, such as FAST
Calculated using the parameters disclosed in this specification and DB) Polypepti
Antibodies that do not bind the enzyme are also included in the invention. The present invention further provides
Under stringent hybridization conditions (as described in
Encoded by a polynucleotide that hybridizes to the light polynucleotide.
Antibodies that bind only to the polypeptide that is used. The antibody of the present invention also has its binding parent.
May be described or specified with respect to compatibility. The preferred binding affinity is the dissociation constant or Kd
But 5X10^-6M, 10^-6M, 5X10^-7M, 10^-7M, 5X10^-8M, 10^-8M, 5X10^-9M, 10^-9M,
5 x 10^-TenM, 10^-TenM, 5X10^-11M, 10^-11M, 5X10^-12M, 10^-12M, 5X10^-13M, 10 ^-13 M, 5X10^-14M, 10^-14M, 5X10^-15M, and 10^-15Including those that are less than or equal to M.

The present invention also relates to purified or isolated antibodies capable of specifically binding a mutant GENSET protein, or a fragment or variant thereof comprising an epitope of a mutant GENSET protein. <Method for Preparing Antibody> The antibody of the present invention can be prepared by a suitable method known in the art. Some of these methods are described in further detail in the examples entitled "Methods for Preparing Antibody Compositions". For example, the polypeptide of the present invention or an antigenic fragment thereof is
It can be administered to animals for the production of serum containing "polyclonal antibodies". As used herein, "monoclonal antibody" is not limited to antibodies produced by hybridoma technology, but rather refers to antibodies derived from a single clone, including eukaryotic, prokaryotic, or phage clones. It does not mean how to produce it. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies. Hybridoma methods include methods known in the art (eg, Harlow et al. 1988;
See Hammerling et al., 1981). (The references above are incorporated by reference in their entirety). Fab and F (ab ') 2 fragments can be produced from hybridoma-producing antibodies by proteolytic cleavage using, for example, papain (which produces the Fab fragment) or pepsin (which produces the F (ab') 2 fragment). . Alternatively, the antibodies of the invention may be produced by the application of recombinant DNA technology or synthetic chemistry using methods known in the art. For example, the antibody of the present invention is
It can be prepared using various phage display methods known in the art. In phage display, functional antibody domains are displayed on the surface of phage particles, which carry the polynucleotide sequences that encode them. Phage with the desired binding properties are selected from a repertoire or combinatorial antibody library (eg, human or mouse) by direct selection with antigen, generally those bound or captured on a solid surface or bead. It Phage used in these methods are generally M13, Fv with fd, and Fab, or by recombinant phage gene II.
Filamentous phage containing a disulfide-stabilized Fv antibody domain fused to I or gene VIII protein. Examples of phage display methods that can be used to generate the antibodies of the invention are described by Brinkman et al., (1995); Ames et al., (1995); Kettleborough, et al.
(1994); Persic, et al. (1997); Burton et al. (1994); PCT / GB91 / 01134; WO 90/02.
809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982
WO 95/20401; and US Patents 5,698,426, 5,223,409, 5,403,484, 5,580,
717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,6
Including those disclosed and disclosed in 37, 5,780,225, 5,658,727 and 5,733,743 (the aforementioned references are incorporated by reference in their entirety).

After phage selection, the antibody coding region from the phage is isolated and used to produce whole antibodies, including human antibodies, or other desired antigen-binding fragments, as described in the above references. , And can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria. For example, techniques for recombinantly producing Fab, Fab 'F (ab) 2 and F (ab') 2 fragments have also been described in WO 92/22324; Mullinax et al. (1992); and Sawai et al. (1995). And Better et al. (1988) (said reference is incorporated by reference in its entirety) and can be used using methods known in the art. Examples of techniques that can be used to produce single chain Fvs and antibodies are described in US Pat.
778 and 5,258,498; Huston et al. (1991); Shu et al. (1993); and Skerra et al.
1988), the entire disclosure of which is incorporated herein by reference. For certain applications, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. Methods for producing chimeric antibodies are known in the art. For example,
Morrison, (1985); Oi et al., (1986); Gillies et al. (1989); and US Patent 5,807,7.
See 15 (the entire disclosure of which is incorporated herein by reference). Antibodies were used for CDR transplantation (EP 0 239 400; WO 91/09967; US Pat. No. 5,530,101; and 5,
585,089), veneer method or refitting method (EP 0 592 106; EP 0 519 596; Padlan, 1
991; Studnicka et al., 1994; Roguska et al., 1994), and chain shuffling (US Pat. No. 5,565,332) may be used for humanization. The entire disclosures of these publications are incorporated herein by reference. Human antibodies can be made by a variety of methods known in the art including the phage display methods described above. For example, further, US Pat. Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; WO 98
/ 46645; WO 98/50433; WO 98/24893; WO 96/34096; WO 96/33735; and WO 9
See 1/10741 (the above reference is incorporated by reference in its entirety). The invention further includes antibodies recombinantly fused or chemically conjugated (including covalent or non-covalent conjugations) to the polypeptides of the invention. The antibody is
There may be specificity for antigens other than the polypeptides of the invention. For example, the antibodies of the invention can be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs or toxins. For example, WO 92/08495; WO 91/14438; WO 89/12624; US Patent 5,314,9
95; and EP 0 396 387. The entire publication is incorporated herein by reference. Fusion antibodies also target the polypeptide of the invention to a particular cell type in vitro or in vivo by fusing or conjugating the polypeptide of the invention to an antibody specific for a particular cell surface receptor. Can be used for Antibodies fused or conjugated to the polypeptides of the invention can also be used in in vitro immunoassays and purifications using methods known in the art (eg,
Harbor et al., Supra; WO 93/21232; EP 0 439 095; Naramura, M. et al., 1994; US Pat. No. 5,474,981; Gillies et al., 1992; Fell et al., 1991). Have been incorporated as).

The invention further includes compositions comprising a polypeptide of the invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptide of the present invention may be the antibody Fc
It can be fused or conjugated to a region, or a portion thereof. The antibody portion fused to a polypeptide of the invention comprises the hinge region, CH1 domain, CH2 domain, and CH3 domain or the entire domain or a combination of portions thereof. The polypeptides of the present invention can be fused or conjugated to the antibody moieties described above using methods known in the art to increase the in vivo half-life of the polypeptide or for use in immunoassays. For example, an Fc portion fused to a polypeptide of the invention can form a dimer due to disulfide bonds between the Fc portions. Polypeptides IgA and IgM
Higher order multimeric forms can be created by fusing with the part of. Methods for fusing or conjugating the polypeptides of the present invention to antibody moieties are known in the art. For example, U.S. Patents 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5
, 112,946; EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashk
See enazi et al. (1991); Zheng et al., (1995); and Vil et al., (1992), which is incorporated by reference in its entirety. Wild-type or transgenic non-human animals or mammals that express different species of GENSET other than those that desire antibody binding, and animals that do not express GENSET (ie, GENSET knockout animals as described herein). Is especially
It is useful for antibody preparation. GENSET knockout animals recognize all or most exposed regions of the GENSET protein as foreign antibodies and thus produce antibodies for a wider array of GENSET epitopes. Furthermore, small polypeptides having only 10-30 amino acids may be useful for obtaining specific binding to one of the GENSET proteins. In addition, the humoral immune system of animals that produce GENSET species that resembles the antigen sequence selectively discriminates between differences between the animal's native GENSET species and the antigen sequence to allow antibodies directed against these unique sites in the antigen sequence. To produce. Such technology is GENSET
It will be particularly useful to obtain antibodies that specifically bind one of the proteins. The antibodies of the present invention can be labeled with one of the radioactive, fluorescent or enzymatic labels known in the art. (Uses of Polynucleotides) Uses of Polynucleotides as Reagents The polynucleotides of the present invention, particularly those described in the section of “oligonucleotide primers and probes”, are used as reagents in isolation methods, diagnostic assays, and forensic methods. Can be used. For example, the sequences from the GENSET polynucleotides of the invention can be detectably labeled and used as probes to isolate other sequences to which they can hybridize. In addition, the sequences from the GENSET polynucleotides of the invention may be used in PCR for isolation, diagnostic, or forensic methods.
Can be used to design primers.

<Forensic Analysis> PCR primers can be used in forensic analysis such as the DNA fingerprinting method described below. Such an analysis may utilize a detectable probe or primer based on the sequence of the polynucleotide of the invention. Accordingly, the invention includes a method of identifying an individual using the polynucleotide of the invention in forensic analysis, the method comprising the steps of: a) obtaining a biological sample containing nucleic acid material from an individual; b ) Obtaining an identification pattern of this individual using the polynucleotides of the invention, in particular GENSET primers and probes; c) comparing said identification pattern with a reference identification pattern; d) said identification pattern is said reference identification pattern Determining if they are the same; in one embodiment of the method, the identification patterns are as described in the sections entitled "Forensic Matching by DNA Sequencing" and "Positive Identification by DNA Sequencing". , Present in the amplicon sequence obtained using the GENSET primer. In other embodiments, the discrimination pattern is obtained using any of the methods described in the sections entitled "Southern blot medical identification,""dot blot identification," and "selective" fingerprint "identification." Present in a unique band or dot pattern. Forensic Matching by DNA Sequencing Method In one example, a DNA sample is isolated by conventional methods from a forensic specimen, eg, hair, semen, blood or skin cells. A panel of PCR primers designed from the different polynucleotides of the invention, using any of the techniques known to those of skill in the art, including those described herein, is then added to about 100 to 100 of forensic specimens. Used to amplify DNA that is 200 bases long. The corresponding sequence is obtained from the test subject. Then, using standard methods, the sequence of each of these discriminating DNAs is determined and a single database comparison is made to determine the difference between the sequence from the control, if any, and the sequence from the sample. A statistically significant difference between the DNA sequence of the suspect and the DNA sequence from the sample conclusively proves the lack of identity. This lack of identity can be demonstrated, for example, by only one sequence. On the other hand, identity should be demonstrated by the fact that multiple sequences all match. Preferably, a statistically identical sequence of at least 50 100 bases long is used to establish identity between the suspect and the sample. [Positive Identification by DNA Sequencing Method] The “forensic matching by DNA sequencing method” technology described in the present specification is
It can be used on a large scale to provide fingerprint identification that is unique to an individual. In this method, primers are prepared from a number of polynucleotides of the invention. Preferably 20-50 different primers are used. These primers are used to obtain a DNA segment that is the corresponding number of PCR products from the individual questioned. Sequencing is performed on each of these DNA segments. The database of sequences produced by this method uniquely identifies the individual who obtained the sequence. The same panel of primers can then be later used to absolutely correlate the tissue or other biological specimen with the individual.

[Southern Blot Medical Identification] The “positive identification by DNA sequencing” described herein is repeated to obtain a panel of at least 10 amplified sequences from individuals and specimens. Preferably the panel comprises at least 50 amplified sequences. More preferably the panel is 10
Contains 0 amplified sequences. In certain embodiments, the panel comprises 200 amplified sequences. The PCR product DNA is then digested with a combination of restriction enzymes specific for 1 or preferably 4 bases. Such enzymes are commercially available and known to those skilled in the art. After digestion, the gene fragments produced are separated by size in multiple replication wells on an agarose gel and transferred to nitrocellulose using Southern blotting techniques known to those skilled in the art. For more information on Southern blotting, see Davis et al. (1986). The entire disclosure of which is incorporated herein by reference. A panel of probes based on the sequences of the polynucleotides of the invention, fragments thereof consisting of at least 10 bases, may be radioactively or colorimetrically labeled using methods known in the art such as nick translation or end labeling, and further Hybridize for Southern blot using methods known in the art. Preferably, the probe comprises at least 12, 15, or 17 contiguous nucleotides from a polynucleotide of the invention. More preferably, the probe comprises at least 20-30 contiguous nucleotides from a polypeptide of the invention. In certain embodiments, the probe comprises 30 or more nucleotides from a polynucleotide of the invention. In other embodiments, the probes are at least 40, at least 50, at least 75, at least 100.
, At least 150 or at least 200 contiguous nucleotides from a polypeptide of the invention. Preferably at least 5-10 of these labeled probes are used, and more preferably about 20-30 are used to provide a unique pattern. The bands that emerge as a result of the hybridization of multiple samples of polynucleotides of the invention are unique identifiers. The banding pattern on the Southern blot is also unique because the restriction enzyme cleavage is different for all individuals. cD
An increase in the number of NA probes statistically leads to a higher confidence level for identification due to an increase in the number of sets of bands used for identification.

Dot Blot Identification Method Another method for identifying an individual using the polynucleotide sequences disclosed herein utilizes the dot blot hybridization method. Genomic DNA is isolated from the nucleus of the subject to be identified. At least 10
About 30 b in length, corresponding to 50, preferably 50 polynucleotide sequences of the invention.
Synthesize p oligonucleotide probe. A probe is used to hybridize genomic DNA with conditions known to those of skill in the art. Oligonucleotides are end-labeled with P ³² using polynucleotide kinase (Pharmacia). The dot blot is prepared by spot-forming the genomic DNA on nitrocellulose or the like using a vacuum dot blot manifold (BioRad, Richmond California). Nitrocellulose filters containing genomic sequences are pre-hybridized with labeled probes and baked or UV-bonded to the filters that have been hybridized (Davis et al., 1986) ³² P labeled DNA fragments using techniques known in the art. , Are sequentially hybridized sequentially under stringent conditions to detect the minimal difference between this 30 bp sequence and DNA. Tetramethylammonium chloride is useful for identifying clones containing a small number of nucleotide mismatches (Wood
1985). The unique pattern of dots distinguishes one individual from another. [Selective "Fingerprint" Identification Method] In a typical selective fingerprinting method, the probe is derived from cDNA. Preferably, multiple probes with sequences from different genes are used as follows. Polynucleotides containing at least 10 contiguous bases from these sequences can be used as probes. Preferably, the probe comprises at least 12, 15, or 17 contiguous nucleotides from the polypeptide of the invention. More preferably, the probe comprises at least 20-30 contiguous nucleotides from a polypeptide of the invention. In certain embodiments, the probe comprises 30 or more nucleotides from a polynucleotide of the invention. In other embodiments, the probe is at least 40,
It comprises at least 50, at least 75, at least 100, at least 150 or at least 200 contiguous nucleotides from a polypeptide of the invention. Oligonucleotides, which are typically 20-mers, can be prepared using commercially available oligonucleotide services such as Genset, Paris, France, e.g. 50, 100 or 2
It is prepared from as many as 00 polynucleotides of the invention. Cell samples from the test subject are processed for DNA using methods known to those of skill in the art. Nucleic acids are digested with restriction enzymes such as EcoRI and XbaI. Following digestion, the sample is added to the wells for electrophoresis. This method known in the art may be modified to accommodate polyacrylamide electrophoresis, but in this example 5 μg
Samples containing DNA are loaded in wells and separated on a 0.8% agarose gel. Gels are transferred onto nitrocellulose using standard Southern blotting techniques.

10 ng of each oligonucleotide is pooled and end labeled with P32. The nitrocellulose is prehybridized with a blocking solution and then hybridized with a labeled probe. After hybridization and washing, the nitrocellulose filter is exposed to X-Omat AR X-ray film. The hybridization pattern generated is unique for each individual. It is further considered in this example that the number of probe sequences used may be varied to obtain additional accuracy or clarity. <Search for corresponding genomic sequence> The GENSET cDNA of the present invention can also be used for cloning a sequence located upstream of the cDNA of the present invention on the corresponding genomic DNA. Such upstream sequences may be capable of modulating gene expression, including promoter sequences, enhancer sequences, and other upstream sequences that affect transcription or translation levels. Once identified and cloned, these upstream control sequences can be used in expression vectors designed to direct expression of the inserted gene in the desired spatial, temporal, developmental, or quantitative manner. [Use of cDNA or fragment thereof for cloning upstream sequence from genomic DNA] The polynucleotide-derived sequence of the present invention can be used to isolate the promoter of the corresponding gene using the chromosome walking method. Genome W available from Glontech
In one chromosomal walking method using the alker (tm) kit, each of 5 samples of complete genomic DNA is digested with different restriction enzymes having 6 base recognition sites to blunt the ends. After digestion, oligonucleotide adapters are attached to each end of the generated genomic DNA fragment. For each of the 5 genomic DNA libraries, the first PCR reaction is performed using the external adapter primer and the external gene-specific primer included in the kit according to the manufacturer's instructions (incorporated herein by reference). The gene-specific primer should be selected to be specific for the polynucleotide of the invention of interest and have a melting point, length, and position in the polynucleotide of the invention that is consistent with its use in the PCR reaction. Should have Each first PCR reaction consisted of 5 ng genomic DNA, 5 μl 10X Tth reaction buffer, 0.2 mM each dNTP, 0.2 μM external adapter primer and 1.1 mM external gene-specific primer in a total volume of 50 μl. Mg (OAc) ₂ , Tth polymerase
Contains 1 μl of 50X mix. The PCR reaction cycle is as follows: 94 ° C (1 minute), 94 ° C (2 seconds), 72 ° C (3 minutes) 7 cycles; 94 ° C (2 seconds), 67 ° C (3 minutes) 32 cycles ); 67 ° C. (5 minutes).

The product of the PCR reaction is diluted and used as a template for the second PCR reaction according to the manufacturer's instructions, with a pair of nested primers resident on the amplicon resulting from the first PCR reaction. . For example, add 5 μl of the reaction product of the first PCR reaction mixture.
It can be diluted 180 times. The reaction is performed in a volume of 50 μl with a composition that is identical to that of the first PCR reaction except using nested primers. The first nested primer is adapter specific and is included in the Genome Walker (tm) kit. The second nested primer should have a melting point, length, and position in the polynucleotide of the invention that is specific to the particular polynucleotide of the invention in which the promoter is cloned and is consistent with its use in the PCR reaction. Is. The reaction parameters for the second PCR reaction are: 94 ° C (1 minute), 94 ° C (2 seconds), 72 ° C (
3 cycles) 6 cycles; 94 ℃ (2 seconds), 67 ℃ (3 minutes) 25 cycles; 67 ℃ (5 minutes). The second PCR reaction product is purified, cloned, and sequenced using standard methods. Alternatively, two or more human genomic DNA libraries can be constructed with two or more restriction enzymes. The digested genomic DNA may be converted into single stranded, circular or linear DNA and cloned into a vector. A biotinylated oligonucleotide containing at least 15 nucleotides from a polynucleotide of the sequence of the invention is hybridized to single-stranded DNA. Hybrids of single-stranded DNA, including biotinylated oligonucleotides and polynucleotides of the invention sequences, are isolated as described herein. After that, single-stranded DNA containing the polynucleotide of the sequence of the present invention is released from the beads, and double-stranded using a primer specific to the polynucleotide of the sequence of the present invention or a primer corresponding to the sequence contained in the cloning vector. Convert to strand DNA. The double-stranded DNA produced is transformed into bacteria. DNA containing the GENSET polynucleotide sequence is identified by colony PCR or colony hybridization. Identification of Promoters in Cloned Upstream Sequences Once the upstream genomic sequence has been cloned and sequenced as described above, the putative promoter and transcription start site within the upstream sequence is located upstream of the polynucleotide of the invention. Sequences can be identified by comparison with databases containing known transcription start sites, transcription factor binding sites or promoter sequences.

Further, the promoter in the upstream sequence can be identified by using a promoter reporter vector as follows. Expression of the reporter gene is detected when placed under the control of a regulatory active polynucleotide fragment or variant, which is the GENSET promoter region located upstream of the first exon of the GENSET gene. Suitable promoter reporter vectors into which the GENSET promoter sequence may be cloned are pSEAP-basic, pSEAP-enhancer, pSEAP-enhancer, commercially available from Clontech.
Includes βgal-basic, pβgal-enhancer, or pEGFP-promoter reporter vectors, or pGL2-basic or pGL3-basic promoterless luciferase reporter gene vectors from Promega. Briefly, each of these promoter reporter vectors has multiple clonings located upstream of a reporter gene that encodes a secreted alkaline phosphatase, luciferase, beta-galactosidase, or an easily assayable protein such as green fluorescent protein. Including the site. The sequence upstream of the GENSET coding region is inserted in either direction into the cloning site upstream of the reporter gene and introduced into a suitable host cell. The level of reporter protein is assayed and compared to the level obtained from the vector lacking the insert at the cloning site. An increase in the expression level in the vector containing the insert compared to the control vector indicates the presence of the promoter in the insert. If desired, the upstream sequence may be cloned into a vector containing an enhancer to increase the level of transcription from the weak promoter sequence. An expression level clearly above the expression observed in the vector lacking the insert indicates the presence of the promoter sequence in the inserted upstream sequence. Promoter sequences in upstream genomic DNA can be further defined by site-directed mutagenesis, linker scanning analysis, or other methods known to those of skill in the art. For example, promoter boundaries can be further investigated by making nested 5'and / or 3'deletions in the upstream DNA using conventional methods such as exonuclease III or appropriate restriction endonuclease digestion. The resulting deletion fragment was inserted into a promoter reporter vector, for example, Coles et al.
1998), it can be determined whether the deletion increases, decreases or reflects promoter activity (the disclosure of which is incorporated herein by reference in its entirety). In this way, the boundaries of the promoter can be defined. If desired, potential discrete regulatory sites within the promoter can be identified using site-directed mutagenesis, linker scanning to eliminate potential transcription factor binding sites within individual promoters or combinations thereof. . The effect of these mutations on the transcription level can be measured by inserting mutations into the cloning site of the promoter reporter vector. Assays of this type are known to those skilled in the art and are described in WO 97/17359, US Patent 5,374,544; EP 582 796; US Patent.
5,698,389; US Patent 5,643,746; US Patent 5,502,176; and US Patent 5,266
, 488, the disclosures of which are incorporated herein by reference in their entireties.

The promoter strength and specificity of each GENSET gene can be assessed by the expression level of a detectable polynucleotide operably linked to the GENSET promoter in different cell types and tissues. The detectable polynucleotide is either a polynucleotide that specifically hybridizes to a predefined oligonucleotide probe, or a polynucleotide that encodes a detectable protein including a GENSET polypeptide or a fragment or variant thereof. possible. Assays of this type are known in the art and are described in US Pat. No. 5,502,176;
And US Pat. No. 5,266,488, the disclosure of which is incorporated herein by reference in its entirety. Some methods are described in further detail elsewhere in this application. Promoters and other control sequences located upstream of the polynucleotides of the invention can be used to design expression vectors that can direct the expression of the inserted gene in the desired spatial, temporal, developmental, or quantitative manner. Promoters that can direct the desired spatial, temporal, developmental, or quantitative patterns can be selected using the results of the expression analysis described herein. For example, if a promoter is desired that confers high expression levels in muscle, a promoter sequence upstream of the polynucleotide of the invention derived from mRNA expressed at high levels in muscle can be used in the expression vector. Such vectors are described in further detail elsewhere in this application. Preferably, the desired promoter is placed near multiple restriction sites to facilitate cloning of the desired insert downstream of the promoter, thus allowing the promoter to drive expression of the inserted gene. The promoter may be inserted into a conventional nucleic acid backbone designed for extrachromosomal replication, integration into the host chromosome or transient expression. Suitable backbones for the present expression vectors include retroviral backbones, eukaryotic episome-derived backbones such as SV40 or bovine papillomavirus, bacterial episome-derived backbones or artificial chromosomes. Preferably, the expression vector also polyA downstream of the multiple restriction sites to direct polyadenylation of mRNA transcribed from the gene inserted into the expression vector.
Including signal.

<Searching for Similar Sequences> The polynucleotide of the present invention has a nucleic acid similar to the polynucleotide of the present invention using any method known to those skilled in the art, including the method based on the hybridization or amplification method described in this section. It can be used for isolation and / or purification. These methods are
Genomic DNA encoding mRNA derived from ET cDNA, mRNA corresponding to GENSET cDNA
Alternatively, it can be used to obtain nucleic acids or fragments thereof that are homologous to GENSET cDNA, such as variants, species homologs, or orthologs. Accordingly, multiple cDNAs that are similar to GENSET polynucleotides may serve as a cDNA library for subsequent evaluation of the encoded protein as described herein or for use in diagnostic assays. The cDNA prepared by any of the methods described therein may be further manipulated to obtain the nucleic acid containing the desired fragment of the cDNA using conventional techniques such as subcloning, PCR, or in vitro oligonucleotide synthesis. . For example, a nucleic acid containing only the coding sequence can be obtained using methods known to those of skill in the art. Similarly, nucleic acids containing other desired fragments of the coding sequence of the encoded protein can be obtained. Indeed, the cDNA of the present invention or a fragment thereof can be used to isolate nucleic acids similar to cDNA from a cDNA library or a genomic DNA library. Such cDNA or genomic DNA libraries may be obtained from commercial sources or described in International Patent Application Publication WO 00/37491, the disclosure of which is incorporated by reference herein in its entirety. Such techniques can be created using techniques known to those of ordinary skill in the art. An example of a method for obtaining a nucleic acid similar to GENSET polynucleotide will be described below. Hybridization-Based Methods Methods for identifying cDNAs in cDNA libraries that hybridize to a given probe sequence are disclosed by Sambrook et al. (1989) and Hames and Higgins (1985), the disclosure of which is fully Are incorporated herein by reference. Similar methods can be used to isolate genomic DNA. Briefly, a cDNA or genomic DNA clone hybridized with a detectable probe is identified and isolated and subjected to the following manipulations: The polynucleotide fragments of the present invention are especially "oligonucleotide primers and probes".
It can be used as the probe defined in the section. A probe containing at least 10 contiguous nucleotides from a GENSET cDNA or fragment thereof is labeled with a detectable label such as a radioisotope or fluorescent molecule. Preferably, the probe comprises at least 12, 15, or 17 contiguous nucleotides from the cDNA or fragment thereof. More preferably, the probe comprises at least 20-30 contiguous nucleotides from the cDNA or fragment thereof. In certain embodiments, the probe comprises 30 or more nucleotides from the cDNA or fragment thereof.

Methods of probe labeling are known and include phosphorylation by polynucleotide kinase, nick translation, in vitro transcription, as well as non-radioactive methods. The cDNA or genomic DNA present in the library is transferred to nitrocellulose or nylon filters for denaturation. After blocking the non-specific sites, the filter is incubated with the labeled probe for a time sufficient for the probe to bind to the cDNA or genomic DNA containing sequences capable of hybridizing. By varying the stringency of the hybridization conditions used to identify a cDNA or genomic DNA that hybridizes to a detectable probe, a cDNA or genome that has different levels of identity to the probe DNA can be identified and isolated as follows. Stringent conditions "Stringent hybridization conditions" are defined as conditions under which only nucleic acids having a high level of identity to a probe can hybridize with the probe. These conditions can be calculated as follows: For probes that are 14-70 nucleotides in length, the melting point (Tm) can be calculated using the following formula: Tm = 81.5 + 16.6 (log (Na +)) + 0.41 (Fraction G + C)-(600 / N), in the formula
N is the length of the probe. If the hybridization is carried out in a solution containing formamide, the melting point can be calculated using the formula: Tm = 81.5 + 16.6 (log (Na +)) + 0.41 (fraction G + C)-(0.
63% formamide)-(600 / N), where N is the length of the probe. Prehybridization is performed using 6X SSC, 5X Denhardt's reagent, 0.5% SDS,
It can be performed in 100 μg denatured fragmented salmon sperm DNA or 6X SSC, 5X Denhardt's reagent, 0.5% SDS, 100 μg denatured fragmented salmon sperm DNA, 50% formamide. Formulations of SSC and Denhardt's solutions are listed in Sambrook et al., 1986. Hybridization is performed by adding the detectable probe to the prehybridization solution listed above. If the probe contains double-stranded DNA, denature it before adding it to the hybridization solution. The filter is contacted with the hybridization solution for a time sufficient for the probe to hybridize to nucleic acids containing sequences that are complementary or homologous. For probes with a length of 200 nucleotides or more, hybridization can be carried out at a temperature of 15-25 ° C or lower than Tm. For short probes such as oligonucleotide probes, hybridization can be carried out at a temperature of 15-25 ° C below Tm. Preferably, for hybridization in 6X SSC, the hybridization is performed at about 68 ° C. Preferably, the hybridization in a solution containing 50% formamide is carried out at about 42 ° C.

After hybridization, the filters are washed in 2X SSC, 0.1% SDS for 15 minutes at room temperature. The filter is then washed with 0.1X SSC, 0.5% SDS for 30 minutes to 1 hour at room temperature. After that, the solution is washed in 0.1X SSC, 0.5% SDS at the hybridization temperature. The final wash is in 0.1X SSC at room temperature. Nucleic acid hybridized to the probe is identified by autoradiography or other conventional method. Changes in stringency for low and moderate conditions hybridization and signal detection
Primarily achieved by manipulating formamide concentration (a low percentage of formamide reduces stringency), salt conditions, or temperature. The above method can therefore be modified to identify nucleic acids with low levels of identity with the probe sequence. For example, the hybridization temperature may be decreased in increments of 5 ° C from 68 ° C to 42 ° C in a hybridization buffer having a sodium concentration of about 1M. After hybridization, the filter is in 2X SSC, 0.5% SDS,
It may be washed at the hybridization temperature. These conditions are "
Conditions of "moderate", below 50 ° C are considered "low" conditions. Alternatively, hybridization may be performed at a temperature of 42 ° C. in a buffer such as 6XSSC containing formamide. In this case, the formamide concentration in the hybridization buffer was adjusted to 50% to identify clones with low levels of identity to the probe.
It may be reduced from 5% to 0% in increments of 5%. After hybridization, the filters may be washed in 6X SSC, 0.5% SDS at 50 ° C. These conditions are considered "moderate" conditions above 25% formamide and "low" conditions below 25% formamide. The cDNA or genomic DNA hybridized to the probe is identified by autoradiography or other conventional method. Changes in the above conditions can be achieved by the inclusion and / or substitution of selective blocking reagents used to suppress the background of hybridization experiments. Common blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercial proprietary formulations. Modifications to the above hybridization conditions may be necessary due to compatibility issues when including certain blocking reagents.

Accordingly, the present invention includes a method for isolating nucleic acids similar to the polynucleotides of the present invention, said method comprising the steps of: a) collecting a collection of cDNA or genomic DNA molecules, SEQ ID NO: 1 At least 12, 15, 18, 20, 23, 25, 28, 30 which is a sequence selected from the group consisting of the sequence of 241, the sequence of the clone insert of the deposited clone pool, and the complementary sequence thereof.
, 35, 40 or 50 contiguous nucleotides and a detectable probe under stringent, moderate or low conditions that hybridize the probe to at least the cDNA or genomic DNA in the collection. B) identifying the cDNA or genomic DNA that hybridizes to the detectable probe; and c) isolating the cDNA or genomic DNA that hybridizes to the probe. [Method by PCR] In addition to the method described above, the GENSET of the present invention is summarized in the following paragraph.
Other protocols can be used to obtain homologous cDNA using the cDNA or fragments thereof. CDNA can be prepared by obtaining mRNA from a tissue, cell, or organism of interest using mRNA preparation methods utilizing poly A selection methods or other methods known to those of skill in the art. A first primer capable of hybridizing to the poly A tail of mRNA is hybridized to mRNA, and a reverse transcription reaction is performed to produce a first cDNA strand. The phrase "capable of hybridizing to the poly A tail of the mRNA" means that the eukaryotic poly (A
) + MRNA, which hybridizes to the 3'end of the mRNA and initiates the synthesis of the first cDNA strand, the so-called oligo (dT) primer, which is meant and includes all primers containing a range of thymidine residues. Methods for producing said oligo (dT) primers and hybridizing them to mRNA to initiate reverse transcription of said hybridizing mRNA to produce a first cDNA strand are known to those skilled in the art and are Current Protocols in Science (Cu
rrent Protocols in Molecular Biology), John Wiley and Sons, Inc. 1997
And Sambrook et al., 1989. The oligo (dT) primer is preferably present in a very excess amount in order to allow the 3'ends of all mRNAs to hybridize with at least one oligo (dT) molecule. The priming and reverse transcription steps are based on the type of reverse transcriptase used,
Preferably, it is carried out at 37 ° C to 55 ° C. Preferred oligo (dT) for initiation of reverse transcription of mRNA
A primer is an oligonucleotide containing a range of thymidine residues of sufficient length to specifically hybridize to the polyA tail of mRNA, preferably 12-18 thymidine residues in length. More preferably, such an oligo (dT) primer comprises an additional sequence upstream of the poly (dT) range, in order to add a given sequence to the 5'end of all first cDNAs, which sequence is It can be used later to facilitate subsequent manipulation of the cDNA. This additional sequence is preferably 8 to 60 residues in length. For example, c
Addition of a regulatory site 5'of the DNA facilitates subcloning of the resulting cDNA. Alternatively, such additional 5'ends can also be used to design PCR primers to specifically amplify the cDNA clone of interest.

The first cDNA strand is then hybridized with the second primer. Any polynucleotide fragment of the invention can be used, but especially those described in the section "Oligonucleotide primers and probes". This second primer comprises at least 10 contiguous nucleotides which is a polynucleotide of the invention. Preferably, the primer comprises at least 10, 12, 15, 17, 18, 20, 23, 25 or 28 contiguous nucleotides, which is a polynucleotide of the invention. In certain embodiments, the primer comprises 30 or more nucleotides that is a polynucleotide of the invention. Contains the complete protein coding sequence, including the true translation start site
If it is desired to obtain a cDNA, the second primer used will contain sequences located upstream of the translation start site. The second primer extends to produce a second cDNA strand that is complementary to the first cDNA strand. Alternatively, RT-PCR may be performed as described above using primers from both ends of the cDNA to be obtained. The double stranded cDNA produced using the method described above is isolated and cloned. The cDNA can be cloned into a vector such as a plasmid or viral vector that is replicable in a suitable host cell. For example, the host cell can be a bacterial, mammalian, avian or insect cell. Isolation of mRNA, production of a first cDNA strand by reverse transcription of a primer hybridized with mRNA, extension of the primer and a second strand complementary to the first cDNA strand
Methods of producing cDNA strands, isolating double-stranded cDNAs and cloning double-stranded cDNAs are well known to those of skill in the art, and current protocols in molecular biology (Current Prot.
ocols in Molecular Biology), John Wiley and Sons, Inc. 1997 and Samb
Explained by rook et al., 1989. Therefore, the present invention includes methods for producing cDNA. In a first embodiment, the method of producing cDNA comprises the following steps: a collection of mRNA molecules from human cells, the group consisting of complementary sequences of SEQ ID NOs: 1-241 and complementary sequences of clone inserts of the deposited clone pool. Contacting with a primer comprising at least 12, 15, 18, 20, 23, 25, 28, 30, 35, 40 or 50 contiguous nucleotides which is a sequence selected from: b) said primer in said collection C) reverse transcribing the hybridization primer to produce a first cDNA strand from the mRNA; d) producing a second cDNA strand that is complementary to the first cDNA strand. And e) isolating the produced cDNA that comprises the first cDNA strand and the second cDNA strand.

Another embodiment of the invention is a purified cDNA obtainable by the method described in the preceding paragraph. In one aspect of this embodiment, the cDNA encodes at least a portion of a human polypeptide. In a second embodiment, the method of producing cDNA comprises the steps of: a) contacting a collection of mRNA molecules from human cells with a first primer capable of hybridizing to the poly A tail of said mRNA; b D) hybridizing said first primer with said poly A tail; c) reverse transcribing said mRNA to produce a first cDNA strand; A sequence selected from the group consisting of at least 12, 15, 18, 20, 23, 25, 28, 30
Creating a second cDNA strand that is complementary to said first cDNA strand using at least one primer comprising 35, 40, or 50 consecutive nucleotides; and e) said first cDNA strand and said Isolating the produced cDNA containing the second cDNA strand. In another aspect of the method, the second cDNA is at least 12, 15 wherein the first cDNA strand is a sequence selected from the group consisting of SEQ ID NOs: 1-241 and sequences of clone inserts of the deposited clone pool. , 18,
Contacting with a second primer comprising 20, 23, 25, 28, 30, 35, 40 or 50 contiguous nucleotides, and a third primer whose sequence is completely contained in the sequence of said first primer; b) performing a first polymerase chain reaction with the second and third primers to produce a first PCR product; c) adding the first PCR product to SEQ ID NOS: 1-241 and a clone insert of the deposited clone pool. At least 12 wherein said sequence is selected from the group consisting of
, A fourth primer comprising 15, 18, 20, 23, 25, 28, 30, 35, 40 or 50 contiguous nucleotides and a fifth primer whose sequence is completely contained in the sequence of said third primer The fourth and fifth primers hybridize to the sequences in the first PCR product; and d) perform a second polymerase chain reaction, thereby producing a second PCR product. To be done.

Alternatively, the second cDNA strand is a sequence that selects the first cDNA strand from the group consisting of SEQ ID NOS: 1-241 and the sequence of the clone insert of the deposited clone pool,
A second primer comprising at least 12, 15, 18, 20, 23, 25, 28, 30, 35, 40 or 50 contiguous nucleotides, and a sequence whose sequence is completely contained in the sequence of said first primer. It can be produced by contacting with three primers and carrying out a polymerase chain reaction with the second and third primers to produce the second cDNA strand. Alternatively, the second cDNA strand is a sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-241 and clone inserts of the deposited clone pool, the first cDNA strand, at least 12, 15, 18,
Contacting with a second primer comprising 20, 23, 25, 28, 30, 35, 40 or 50 consecutive nucleotides; b) hybridizing said second primer with said first strand cDNA; and c) The second hybridization primer can be extended to produce the second cDNA strand. Another embodiment of the present invention is a purified cDNA obtainable by the cDNA production method of the present invention. In one aspect of this embodiment, the cDNA encodes at least a portion of a human polypeptide. [Other protocols] Alternatively, other methods may be used to obtain homologous cDNA. In one method, cDNA is prepared from mRNA and cloned into double-stranded phagemid as follows. The cDNA library in the double-stranded phagemid is then made single-stranded by treatment with endonucleases and exonucleases such as the gene II product of phage F1 (Chang et al., 1993, the entire disclosure of which is incorporated herein by reference). Have been incorporated as). A biotinylated oligonucleotide containing a fragment sequence that is a known GENSET cDNA, genomic DNA or a fragment thereof is hybridized to a single-stranded phagemid. Preferably, the fragment is at least 10, 12, 15, 17, 20, 23, 25, or a sequence selected from the group consisting of the sequences SEQ ID NOs: 1-241 and the sequences of the clone inserts of the deposited clone pool. It contains 28 consecutive nucleotides.

Hybrids between biotinylated oligonucleotides and phagemids are isolated by incubating the hybrids with streptavidin-coated paramagnetic beads and collecting the beads with a magnet (Fry et al., 1992, the disclosure of which is fully incorporated by reference). Incorporated herein by reference). The resulting phagemid is then released from the beads and converted to double-stranded DNA using primers specific for the GENSET cDNA or fragment used to design the biotinylated oligonucleotide. Alternatively, a protocol such as the Gene Trapper kit (Gibco BRL), the disclosure of which is incorporated herein by reference in its entirety, may be used. The double-stranded DNA produced is transformed into bacteria. A cDNA homologous to the CENSET cDNA or a sequence fragment thereof is identified by colony PCR or colony hybridization. <As a Chromosomal Marker> The location of the cDNA of the present invention on the chromosome was determined using information from public and trademarked databases. Table VIII lists the putative chromosomal locations of the polynucleotides of the invention. Column 1 lists the SEQ ID NOs and the corresponding chromosome positions are listed in column 2. Accordingly, the invention also employs the chromosomal location of the polynucleotides of the invention to construct a human high resolution map, or using techniques known to those of skill in the art, including those disclosed below, to determine whether a given sample is present in a sample. To methods and compositions for identifying the chromosomes of GENSET polynucleotides are also described in Radiation Hybrid (RH) Mapping, PCR Mapping and Fluorescence In Situ Hybridization (described below).
The chromosomal location can be mapped using methods or techniques known to those of skill in the art, including FISH). Radiation Hybrid Mapping Radiation hybrid (RH) mapping is a somatic genetic method that can be used for high resolution mapping of the human genome. In this method, a cell line containing one or more human chromosomes is lethally irradiated to cleave each chromosome into fragments of dose-dependent size. These fragments are rescued by fusion with cultured rodent cells to produce subclones containing different fragments of the human genome. This method is described by Benham et al., (1989) and Cox et al., (1990), the disclosure of which is incorporated herein by reference in its entirety. The random and independent nature of subclones allows efficient mapping of human genomic markers. Human DNA isolated from a panel of 80-100 cell lines is GENSET
Provides mapping reagents for ordering cDNA or genomic DNA. In this method, the frequency of cleavage between markers is used to measure distance, as is done with the conventional EST (Schuler et al., 1996, the disclosure of which is incorporated herein by reference in its entirety). It enables the construction of excellent resolution maps.

RH mapping is based on growth hormone (GH) and thymidine kinase (TK) (Foster et al.,
1996) human chromosome 17q22-q25.3, the region surrounding the Gorlin syndrome gene (Obermayr et al., 1996), 60 loci governing the entire short arm of chromosome 12 (Raeymaekers et al., 1995), neurofibroma. The human chromosome 22 region containing the type 2 locus (Frazer et al., 1992) and 13 loci on the long arm of chromosome 5 (W
have been used to generate high resolution whole genome radiation hybrid maps for Arrington et al., 1991), the disclosures of which are incorporated herein by reference in their entireties. [CDNA Mapping to Human Chromosome Using PCR Method] GENSET cDNA and genomic DNA can be assigned to a human chromosome using a PCR-based methodology. In such methods, oligonucleotide primer pairs are designed from the cDNA sequences to minimize the chance of being amplified by the intron.
Preferably, the oligonucleotide primers are 18-23 bp in length and are designed for PCR amplification. Generation of PCR primers from known sequences is known to those of skill in the art. For PCR technology, see Erlich (1992). The entire disclosure of which is incorporated herein by reference. The primers are used in the polymerase chain reaction (PCR) to amplify the template from total human genomic DNA. PCR conditions are as follows: 60 ng of genomic DNA is PCR
As template, 80 ng of each oligonucleotide primer, 0.6 units of Taq polymerase, and 1 uCu of 32 P-labeled deoxycytidine triphosphate are used. P
CR is performed in a microplate thermocycler (Techne) under the following conditions. 30 cycles of 94 ° C (1.4 minutes), 55 ° C (2 minutes) and 72 ° C (2 minutes) with a final extension of 72
° C (10 minutes). Amplification products were analyzed on a 6% polyacrylamide sequencing gel,
Observe by autoradiography. If the length of the generated PCR product is the same as the distance between the ends of the primer sequence in the cDNA from which the primer is derived, then human
-Two panels of rodent somatic cell hybrids, BIOS PCRable DNA (BIOS Corporati
on) and NIGMS human-rodent somatic cell hybrid mapping panel number 1 (NI
The PCR reaction is repeated with a DNA template from GMS, Camden, NJ). PCR is used to screen a series of somatic cell hybrid cell lines containing a set of human chromosomes defined for the presence of a given cDNA or genomic DNA.
DNA is isolated from somatic cell hybrids and used as the starting template in a PCR reaction with a primer pair derived from GENSET cDNA or genomic DNA. Only somatic cell hybrids having a chromosome containing the human gene corresponding to the GENSET cDNA or genomic DNA produce the amplified fragment. GENSET cDNA or genomic DNA is assigned to a chromosome by analysis of the segregation pattern of PCR products derived from somatic hybrid DNA templates. One human chromosome present in all cell hybrids producing the amplified fragment is the chromosome containing the GENSET cDNA or genomic DNA. For methods of somatic gene mapping experiments and analysis of results, see Ledbetter et al., (1990), the disclosure of which is incorporated herein by reference in its entirety.

CDNA Mapping to Chromosomes Using Fluorescent In Situ Hybridization Fluorescent in situ hybridization allows GENSET cDNA or genomic DNA to be mapped to specific locations on a given chromosome. The chromosomes used in the fluorescence in situ hybridization method can be obtained from a variety of sources including cell culture, tissue, or whole blood. In a preferred embodiment, the chromosomal location of the GENSET cDNA or genomic DNA is C
Acquired by FISH as described by herif et al. (1990). The entire disclosure of which is incorporated herein by reference. Metaphase chromosomes are prepared from phytohemagglutinin (PHA) stimulated blood cell donors. PHA from a healthy man
Stimulated lymphocytes are cultured for 72 hours in RPMI-1640 medium. For synchronization, methotrexate (10 uM) was added for 17 hours, followed by 5-bromodeoxyuridine (5-BudR).
, 0.1 mM) for 6 hours. Colcemid (1 u
g / ml). Cells were harvested, washed in RPMI, and in KCl (75 mM) hypotonic solution,
Incubate at 37 ° C for 15 minutes and fix in a solution with 3 changes of methanol: acetic acid (3: 1). The cell suspension is dropped on a glass slide and air dried. GENS
ET cDNA or genomic DNA can be obtained from the manufacturer (Bethesda Research Laboratories, Beths
Esda, MD), labeled with biotin-16dUTP by nick translation, purified using a Sephadex G-50 column (Pharmacia, Upssala, Sweden) and then precipitated by nick translation. Immediately prior to hybridization, dissolve the DNA pellet in hybridization buffer (50% formamide, 2X SSC, 10% dextran sulfate, 1 mg / ml sonicated salmon sperm DNA, pH 7) and probe at 70 ° C. In 5 to 1
Denaturate for 0 minutes. Slides kept at -20 ° C are treated with RNAse A (100 ug / ml) at 37 ° C for 1 hour, then rinsed 3 times in 2X SSC and denatured in the ethanol series.
Chromosome preparations were denatured in 70% formamide, 2 X SSC at 70 ° C for 2 minutes, then
Dehydrate at 4 ° C. Slides should have the protease K (20 mM Tris-HCl, 2 mM
Add 10 ug / 100 ml of calcium chloride), treat at 37 ° C for 8 minutes, and dehydrate.
The hybridization mixture containing the probe is placed on a slide, covered with a coverslip, sealed with a rubber paste, and then incubated overnight at 37 ° C. in a humidified container. After hybridization and post-hybridization washes, the biotinylated probe is detected by avidin-FITC and amplified with the addition of additional layers of biotinylated goat anti-avidin and avidin-FITC. For chromosomal location, a fluorescent R band is obtained as previously described (Cherif et al., 1990). Observe slides under LEICA fluorescence microscope (DMRXA). The chromosomes are counterstained with propidium iodide and the fluorescent signal of the probe appears as two symmetrical yellow-green spots on both chromatides of the fluorescent R-band chromosome (red). Therefore, the specific GENSE
T cDNA or genomic DNA can be localized to a particular cytogenetic R band on a given chromosome.

Uses of cDNA for Construction or Expansion of Chromosome Maps Once GENSET cDNA or genomic DNA has been assigned to a particular chromosome using methods known to those of skill in the art, particularly as described herein. Once constructed, a high resolution map of the chromosomes in which they are located can be constructed or used to identify the chromosomes in the sample. Chromosome mapping involves assigning certain unique sequences to particular chromosomes, as described above. Once the unique sequence is mapped to a given chromosome, it is ordered in relation to other unique sequences located on the same chromosome. One method of chromosome mapping utilizes a set of yeast artificial chromosomes (YACs) that carry thousands of inserts derived from the chromosome of the organism from which the GENSET cDNA or genomic DNA was obtained. This method is described in Nagaraja et al. (1997), the disclosure of which is incorporated herein by reference in its entirety. Briefly, in this method, each chromosome is cut into overlapping fragments and inserted into the YAC vector. The YAC insert is screened using PCR or other methods to determine if its position contains the GENSET cDNA or genomic DNA to be determined. Once gen
When an insert containing a SET cDNA or genomic DNA is found, it is known that the insert is located further on the chromosome, or on the region where the GENSET cDNA or genomic DNA is derived, by analysis by PCR or other method. It is determined whether another sequence is further included. This process is repeated to locate each GENSET cDNA or genomic DNA for each insert in the YAC library, relative to each other and other known chromosomal markers. In this way, a high resolution map of the distribution of multiple unique markers for each of the biochromosomes can be obtained. Identification of Genes Associated with Genetic Diseases or Drug Responses This example illustrates a method useful for associating GENSET cDNA or genomic DNA with specific phenotypic features. In this example, a particular GENSET cDNA or genomic DNA is used as a test probe to associate a particular phenotypic trait with a GENSET cDNA or genomic DNA. The GENSET cDNA or genomic DNA is mapped to specific locations on the human chromosome using methods such as those described herein or other methods known in the art. Search for Mendelian inheritance in humans (V. McKusick, Mendelian Inheritance in Man_ (Johns Hopkins Unive
(available online from the rsity Welch Medical Library) is a region of human chromosomes containing GENSET cDNA or genomic DNA that is extremely gene-concentrated to contain several known genes and some diseases or phenotypes for which no genes have been identified. It is revealed that the area is high. The gene corresponding to this GENSET cDNA or genomic DNA is therefore a direct candidate for each of these genetic disorders.

Cells from patients with these diseases or phenotypes are isolated and grown in culture.
Screen genomic DNA, mRNA, or cDNA obtained from the patient using GENSET cDNA or PCR primers derived from genomic DNA. GENSET c not amplified in patient
DNA or genomic DNA may be clearly associated with a particular disease by further analysis. Alternatively, PCR analysis may produce fragments of different lengths if the sample is from an individual with a phenotype associated with the disease rather than from a healthy individual, and the gene containing the cDNA is Indicates that it may be the cause of a genetic disorder. Uses of Polynucleotides in Recombinant Vectors The present invention also includes a recombinant vector comprising an isolated polynucleotide of the invention or a fragment thereof, and a host cell that is recombinant for a polynucleotide of the invention such as the above vectors, and Such vectors and methods of making host cells and methods of using them by recombinant techniques to produce GENSET polypeptides are related. <Recombinant vector> “Vector”, as used herein, is a double-stranded or single-stranded,
A circular or linear DNA or RNA molecule containing at least one polynucleotide of interest that is desired to be delivered to a cellular host or a single cell or multicellular host organism. The present invention includes a family of recombinant vectors containing regulatory and / or coding polynucleotides derived from either the GENSET genomic sequence or the cDNA sequence. Generally, the recombinant vectors of the present invention include any of the polynucleotides described herein, including regulatory sequences, coding sequences and polynucleotide constructs, as well as GENSET primers or probes as defined herein. Can also include. In a first preferred embodiment, the recombinant vector of the invention is used to amplify an inserted polynucleotide derived from a GENSET genomic sequence or GENSET cDNA, for example SEQ ID NO: 1 in a suitable cell host. ~ 241, a cDNA clone selected from the group consisting of the sequences of the clone insert of the deposited clone pool, variants and fragments thereof, the polypeptide being amplified whenever the recombinant vector replicates.

A second preferred embodiment of the recombinant vector according to the present invention comprises an expression vector comprising a regulatory polynucleotide or a coding nucleic acid according to the present invention or both.
In certain embodiments, the expression vector is used to express a GENSET polypeptide that can be subsequently purified, and further used, for example, in a ligand screening assay or to produce specific antibodies directed against the GENSET protein. Used as a raw material. In other embodiments, expression vectors are used in the generation of transgenic animals, as well as gene therapy. Expression must be provided with appropriate signals in the vector, said signals including various regulatory elements such as enhancers / promoters from both viral and mammalian sources which cause expression of the gene of interest in the host cell. . The dominant drug selectable marker for establishing permanent, stable cell clones expressing the product is generally the vector of the invention because the expression of the drug selectable marker is an element associated with expression of the polypeptide. Is contained in the expression vector of the present invention. More particularly, the present invention relates to GENSET proteins, preferably SEQ ID NOS: 242-4.
Amino acid sequence selected from the group consisting of 82 sequences, SEQ ID NOs: 242-272 and 274
To 384 sequences, as well as a GENSET protein having the mature protein sequence encoded by the clone insert in the deposited clone pool, and the GENSET protein having variants and fragments thereof. The polynucleotides of the invention can be used to express the encoded protein in a host organism and produce a beneficial effect. In such a method, the encoded protein may be expressed transiently in the host organism or may be stably expressed in the host organism. The encoded protein can have any of the activities described herein. The encoded protein is a protein that the host organism lacks, or the encoded protein may enhance existing protein levels in the host organism. Some of the elements found in the vectors of the invention are described in detail in the sections below. [General characteristics of the expression vector of the present invention] The recombinant vector according to the present invention includes YAC (yeast artificial chromosome), BAC (bacterial artificial chromosome), phage, phagemid, cosmid, plasmid, or chromosomal, non-chromosomal, semi-synthetic and Includes, but is not limited to, linear DNA molecules that include synthetic DNA. Such recombinant vectors may include transcription units that include the following assemblies: (1) Genetic element (s) that have a regulatory role in gene expression, such as promoters or enhancers. Enhancers are generally cis-300 bp in length and are cis-acting elements of DNA that act on a promoter to increase transcription.

(2) A structure or coding sequence that is transcribed into mRNA and ultimately translated into a polypeptide, wherein said structure or coding sequence is operably linked to the regulatory elements described in (1). And (3) appropriate transcription start and end sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably contain a leader sequence enabling extracellular secretion of translated protein by the host cell. Alternatively, the recombinant protein may include an N-terminal residue if expressed without a leader or transport sequence. This residue may or may not be cleaved from the subsequently expressed recombinant protein to yield the final product. In general, recombinant expression vectors contain an origin of replication, a selectable marker that enables transformation of the host cell, and a promoter derived from the highly expressed gene that directs the transcription of downstream structural sequences. Including. Heterologous structural sequences are constructed at appropriate stages by translation initiation and termination sequences, preferably a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. . In certain embodiments where the vector is applied to transfect and express desired sequences in mammalian host cells, preferred vectors will include an origin of replication in the desired host, appropriate promoters and enhancers, and also Ribosome binding sites, polyadenylation signals, splice donor and acceptor sites, transcription termination sequences, and 5'non-transcribed sequences. For example, SV40 origin SV40
In DNA sequences derived from the viral genome, early promoters, enhancers, splices and polyadenylation signals can be used to supply the required nontranscribed genetic elements. In vivo expression of the GENSET polypeptides of the present invention may be useful for expression of unchanged genes in the host organism or for correction of genetic defects associated with the production of bioinactive GENSET protein. Thus, the invention also includes a recombinant expression vector designed primarily for the in vivo production of the GENSET polypeptides of the invention by introducing appropriate genetic material into the organism to be treated or into the patient. This genetic material can be introduced in vitro into cells previously extracted from the organism, and the modified cells can subsequently be reintroduced into the organism and introduced in vivo directly into the appropriate tissue.

Control Elements Suitable promoter regions for use in the expression vectors according to the invention are selected taking into account the cell host in which the heterologous gene has to be expressed. The particular promoter used to control the expression of the nucleic acid sequence of interest is not considered critical so long as it is capable of directing the expression of the nucleic acid in the target cell. Thus, where human cells are the target, the nucleic acid coding region is placed adjacent to and under the control of a promoter capable of being expressed in human cells, such as, for example, human or viral promoters. It is preferable. Suitable promoters can be heterologous with respect to the nucleic acid of interest controlling expression, or endogenous to the unaltered polynucleotide containing the coding sequence to be expressed. Moreover, promoters are generally heterologous with respect to the recombinant vector sequence into which the construct promoter / coding sequence is inserted. The promoter region can be selected from the desired gene using, for example, the CAT (chloramphenicol transferase) vector, and more preferably the pKK232-8 and pCM7 vectors. Preferred bacterial promoters are LacI, LacZ, T3 or T7 bacteriophage R
NA polymerase promoter, gpt, lambda PR, PL and trp promoter (EP 0036776), polyhedrin promoter, or baculovirus-derived p
10 protein promoter (Kit Novagen) (Smith et al., 1983; O'Reilly et al., 199)
2, the disclosure of which is incorporated herein by reference in its entirety), the lambda PR promoter or even the trc promoter. Eukaryotic promoters include early CMV, HSV thymidine kinase, early and late
Includes SV40, LTR from retrovirus, and mouse metalthionein-L. Selection of convenient vectors and promoters is within the skill of the art. Selection of promoters is within the skill of one of ordinary skill in the genetic engineering art. For example, Sambrook
(1989) or the methods described in Fuller et al. (1996), the disclosures of which are incorporated herein by reference in their entireties. When using other regulatory element cDNA inserts, it is generally desirable to include a polyadenylation signal to affect the correct polyadenylation of gene transcription. The nature of the polyadenylation signal is not believed to be critical to the successful practice of the present invention and any such sequence may be used for human growth hormone and the SV40 polyadenylation signal. Also planned as elements of the expression cassette are terminators. These elements serve to enhance message levels and minimize readthrough from the cassette to other sequences.

Selectable Markers Selectable markers provide identifiable changes to cells so that the cells containing the expression construct can be readily identified. The selectable marker gene for selection of transformed host cells is
Preferably, it is dihydrofolate reductase or neomycin resistance to eukaryotic cultured cells, TRP1 to S. cerevisiae or tetracycline, rifampicin or ampicillin resistance in E. Coli (E. coli) or Levan saccharase of actinomycete, and this last The marker is a negative selection marker. Preferred Vectors Bacterial Vectors Representative, but not limiting, useful expression vectors for bacterial use include:
It may include a bacterial origin of replication derived from a commercially available plasmid containing the selectable marker and the genetic elements of pBR322 (ATCC 37017). Such commercially available vectors are, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and pGEM1 (Promega Biotec,
Madison, WI, USA). Many other suitable vectors are known to those of skill in the art and are commercially available, such as the following bacterial vectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10.
, Phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18
A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5
(Pharmacia); pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3
, PBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIAexpress). Bacteriophage Vectors P1 bacteriophage vectors can contain large inserts ranging from about 80 to about 100 kb. Construction of P1 bacteriophage vectors such as p158 or p158 / neo8 is described primarily by Sternberg (1992, 1994), the disclosure of which is incorporated herein by reference in its entirety. Recombinant P1 clones containing the GENSET nucleotide sequence can be designed to insert large polynucleotides of 40 kb or larger (See Linton et al., 1993), the disclosure of which is incorporated herein by reference in its entirety. A preferred protocol for producing P1 DNA for transgenic experiments is the protocol described by McCormick et al. (1994),
The entire disclosure of which is incorporated herein by reference. Briefly, E. coli incorporating the P1 plasmid (preferably NS3529 strain) is grown overnight in a suitable broth medium containing 25 μg / ml kanamycin. P1 DNA is prepared from E. coli by alkaline lysis using the Qiagen Plasmid Maxi Kit (Qiagen, Chatsworth, CA, USA) according to the manufacturer's instructions. Purify P1 DNA from the lysate on two Qiagen-tip 500 columns using the wash and elution buffers included in the kit. Perform a phenol / chloroform extraction before precipitating the DNA with 70% ethanol. After solubilizing the DNA in TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA), DN
A concentration is determined by spectroscopic measurement.

If the aim is to express a P1 clone containing the GENSET nucleotide sequence in a transgenic animal, generally a transgenic mouse, then for example in the rare P1 polylinker (SfiI, NotI or SalI) It is desirable to remove the vector sequence from the P1 DNA fragment by cleaving the P1 DNA at the cleavage site. The P1 insert is then used using a method similar to the method originally reported for DNA isolation from YAC (see, eg, Schedl et al., 1993a; Peterson et al., 1993. The disclosures of these are fully incorporated herein. (Incorporated by reference), and purified from the vector sequence on a pulse field agarose gel. At this stage, the purified DNA inserts generated can be transferred to Millipore Ultrafree-MC filter units (
Microinjection with 100 mM sodium chloride, 30 μM spermine, 70 μM spermidine (Type VS, 0.025 μM Millipore) on a microdialysis membrane, followed by concentration over Millipore, Bedford, MA, USA, 30,000 molecular weight limit). Dialyze against a buffer (10 mM Tris-HCl, pH 7.4; 250 μM EDTA). The integrity of the purified P1 DNA insert was confirmed by 1% agarose (Sea Kem GTG;
FMC Bio-products) Judged by electrophoresis on a pulse field gel and staining with ethidium bromide. Viral Vectors In one particular embodiment, the vector is derived from an adenovirus. Preferred adenovirus vectors according to the present invention are those described by Feldman and Steg (1996) or Ohno et al. (1994), the disclosure of which is incorporated herein by reference in its entirety. Other preferred recombinant adenoviruses according to this particular embodiment of the invention are human adenovirus type 2 or 5 (Ad2 or Ad5) or adenovirus of animal origin (French patent application no. FR-93.05954), published Is incorporated herein by reference in its entirety. Retroviral vectors and adeno-associated viral vectors are generally
Understood as a recombinant gene delivery system of choice for in vivo delivery of exogenous polynucleotides, particularly to mammals, including humans. These vectors provide efficient gene delivery into cells and the transferred nucleic acid is
Stable integration into host chromosomal DNA. Particularly preferred retroviruses for the preparation or construction of the retroviral in vitro or in vitro gene delivery vehicles of the present invention include mink cell focus inducing virus, mouse sarcoma virus,
Includes reticuloendotheliosis virus and Rous sarcoma virus. Particularly preferred murine leukemia viruses are the 4070A and 1504A viruses, Abelson (ATCC No. VR-
999), friend (ATCC number VR-245), gross (ATCC number VR-590), Rauscher (ATCC No VR-998) and Moloney murine leukocyte virus (ATCC).
No. VR-190; International Patent Application Publication WO 94/24298). Particularly preferred Rous sarcoma virus comprises Brian high titers (ATCC numbers VR-334, VR-657, VR-726, VR-659 and VR-728). Other preferred retroviral vectors are Roth et al. (19
96), International Patent Application Publication WO 93/25234, International Patent Application Publication WO 94/06920, Roux
Et al. (1989), Julan et al. (1992) and Neda et al. (1991), the disclosure of which is incorporated herein by reference in its entirety.

Yet another viral vector system contemplated by the present invention comprises adeno-associated virus (AAV). Adeno-associated viruses require other viruses, such as adenovirus or herpes virus, as helper viruses for efficient replication and the productive life cycle (Muzyczka et al., 1992, the disclosure of which is incorporated herein in its entirety). Is incorporated as a reference). It is one of the few viruses that can integrate DNA into non-dividing cells and exhibits a high frequency of stable integration (
Flotte et al. 1992; Samulski et al., 1989; McLaughlin et al., 1989, the disclosures of which are incorporated herein by reference in their entirety). One advantageous feature of AAV derives from its low potency on transduced primary cells compared to transformed cells. BAC Vector The entire disclosure of which is incorporated herein by reference, the bacterial artificial chromosome (BAC) cloning system (Shizuya et al., 1992) is a large fragment of genomic DNA (100-300 kb) in E. coli. Developed to maintain stability. Preferred BAC vectors include the pBeloBAC11 vector described by Kim et al. (1996), the disclosure of which is incorporated herein by reference in its entirety. The BAC library prepares this vector with size-selected genomic DNA partially digested with an enzyme that allows binding to the Bam HI or Hind III site in the vector. These flanking cloning sites are T7 and SP6 RNA polymerase transcription start sites that can be used to produce final probes by RNA transcription or PCR methods. After preparation of the BAC library in E. coli, BAC DNA is purified from the host cells as a cycle of supercoiling. These cyclic molecules are converted to a linear form prior to size determination and introduction of BAC into recipient cells. The cloning site is flanked by two Not I sites and thus the Not I of the cloned segment.
Allows cleavage from the vector by digestion. Alternatively, the DNA insert contained in the pBeloBAC11 vector is a commercially available enzyme lambd that induces cleavage at a unique cosN site.
It is possible to linearize by treating the BAC vector with a terminase (terminating enzyme).
BAC clone is generated. Baculovirus: Another particularly suitable host vector system is the SF9 cell line from Spodoptera frugiperda (
PVL1392 / 1393 baculovirus transfer vector (Pharmingen) used to transfect ATCC number CRL 1711). Other suitable vectors for expression of GENSET polypeptides of the invention in baculovirus expression systems include those described by Chai et al., (1993), Vlasak et al., (1983) and Lenhard et al., (1996). , The entire disclosure of which is incorporated herein by reference.

Delivery of Recombinant Vectors In order to affect the expression of the polynucleotides and polynucleotide constructs of the invention, these constructs must be delivered intracellularly. This delivery can be accomplished in vitro as in experimental methods for transformation of cell lines, or in vivo or ex vivo as in the treatment of certain disease states. One mechanism is viral infection in which the expression construct is encapsulated within infectious viral particles. Several non-viral methods for the delivery of polynucleotides into cultured mammalian cells have also been designed by the present invention, the calcium phosphate precipitation method (Graham
Et al., 1973; Chen et al., 1987); DEAE-dextran method (Gopal, 1985); electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984); direct microinjection method (Harland et al., 1985); DNA loading. Liposomes (Nicolau et al., 1982; Fraley et al., 1979); and receptor-mediated transfection (Wu and Wu, 1987, 1988).
), But not limited thereto, the disclosures of which are incorporated herein by reference in their entireties. Some of these methods are well adapted for in vivo or ex vivo applications. Once the expressed polynucleotide is delivered into the cell, it stably integrates into the genome of the recipient cell. This integration can be cognate position and orientation by homologous recombination (gene replacement) or random integration at non-specific positions (gene enhancement). In yet another embodiment, the nucleic acids are
It can be stably maintained as an episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences that are sufficient to allow maintenance and replication independent of, or in synchronization with, the host cell cycle. One particular embodiment for a method for delivering a protein or peptide to the interior of a vertebrate cell in vivo is to provide a physiologically acceptable carrier and a naked polypeptide that operatively encodes the polypeptide of interest. Of the polynucleotide of the invention is introduced into the interstitial space of the tissue containing the cells, whereby the naked polynucleotide is internalized inside the cell and has a physiological effect. It is particularly adaptable for in vitro transmission, but may be applicable in vivo as well. Compositions for in vitro and in vivo applications containing "naked" polynucleotides are described in International Patent Application Publication WO 90/11092 (Vical Inc.) and also International Patent Application Publication WO 95/11307 (Institut Pasteur, INSERM, Universite. d'Ottawa) and Tacson et al. (1996) and Huygen et al. (1996), the disclosures of which are incorporated herein by reference in their entirety.

Another embodiment of the invention further comprises a polynucleotide construct of the invention,
Delivery of naked polynucleotides of the invention into cells may be caused by particle biolistic, as described by Klein et al. (1987), wherein the particles are DNA-coated particle guns and It is capable of accelerating to penetrate the cell membrane and enter the cell without killing it (the disclosure of which is incorporated by reference in its entirety). In a further embodiment, the polynucleotides of the invention are entrapped within liposomes (Ghosh and Bacchawat, 1991; Wong et al., 1980; Nicolau et al., 1987). The entire disclosures of which are incorporated herein by reference. In certain embodiments, the invention provides compositions for the in vivo production of GENSET proteins or polypeptides described herein. The composition operatively encodes the polypeptide in solution in a physiologically tolerable carrier and is introduced into tissue for expression of the protein or polypeptide in tissue cells. A naked polynucleotide that is suitable for. The amount of vector to be injected into the desired host organism will vary depending on the site of injection.
As an indicated dose, 0 in the animal body, preferably in the mammalian body, for example in the mouse body.
1-100 μg of vector is injected. In another embodiment of the vector according to the invention, it can be introduced in vitro into a host cell, preferably a host cell previously harvested from the animal to be treated, more preferably a somatic cell such as a muscle cell. In a subsequent step, cells transformed with a vector encoding the desired GENSET polypeptide or a desired fragment thereof are reintroduced into the animal body to deliver the recombinant protein locally or systemically in the body. Secretion Vectors Certain GENSET cDNAs or genomic DNAs of the invention can also be used to make secretion vectors capable of directing the secretion of the protein encoded by the gene inserted into the vector. Such a secretory vector may facilitate purification or enrichment of the protein encoded by the inserted gene by reducing the number of background proteins in which the desired protein must be purified or enriched. A typical secretion vector is described below.

The secretory vector of the present invention contains a promoter capable of directing gene expression in the intended host cell, tissue or organism. Such promoters include the Rous sarcoma virus promoter, SV40 promoter, human cytomegalovirus promoter, and other promoters known to those of skill in the art. A signal sequence from the polynucleotide of the invention, preferably SEQ ID NOs: 1-31.
And a signal sequence selected from the group consisting of the signal sequence of 33-143 and the sequence of the clone insert of the deposited clone pool is transcribed from the promoter.
The mRNA is operably linked to a promoter to direct translation of the signal peptide. A host cell, tissue, or organism is any cell that recognizes the signal peptide encoded by the signal sequence in the GENSET cDNA or genomic DNA,
It may be a tissue or an organism. Suitable hosts include mammalian cells, tissues or organisms, avian cells, tissues or organisms, insect cells, tissues or organisms, or yeast. In addition, the secretory vector contains cloning sites for insertion of the gene encoding the secreted protein. The cloning site has a signal peptide
It facilitates the cloning of the inserted gene in frame with the signal sequence so that a fusion protein fused to the protein encoded by the inserted gene will be expressed from the mRNA transcribed from the promoter. The signal peptide directs extracellular secretion of the fusion protein. The secretory vector may be DNA or RNA, is integrated into the host chromosome, is stably maintained as an extrachromosomal replicon in the host, is an artificial chromosome, and may be transiently present in the host. Preferably, the secretory vector is maintained in multiple copies within each host cell. As used herein, multiple copies means at least 2, 5, 10, 20, 25, 50 or 50 or more copies per cell. In certain embodiments, multiple copies are maintained extrachromosomally. In other embodiments, the multiple copies result from amplification of a chromosomal sequence. Numerous nucleic acid backbones suitable for use as secretory vectors are known to those of skill in the art and include retroviral vectors, SV40 vectors, bovine papillomavirus vectors, yeast integrative plasmids, yeast episomal plasmids, yeast artificial chromosomes, human artificial chromosomes, P element vectors, baculoviruses, or bacterial plasmids that can be transiently introduced into the host.

The secretory vector also contains a poly A signal such that the poly A signal is located downstream of the gene inserted into the secretory vector. After inserting a gene encoding a protein desired to be secreted into a secretion vector, the secretion vector is subjected to calcium phosphate precipitation method, DEAE-dextran method, electroporation method,
Used as liposomal lipid-mediated transfection, viral particles or naked DNA to introduce into host cells, tissues, or organisms. The protein encoded by the inserted gene is then subjected to ammonium sulfate precipitation, immunoprecipitation, immunochromatography, size exclusion chromatography, ion exchange chromatography, and
Purified or concentrated from the supernatant using conventional techniques such as hplc. Alternatively,
The secreted protein is fully concentrated or pure in the supernatant or in the growth medium of the host and may be used for its intended purpose without further concentration. The signal sequence may also be inserted in a vector designed for gene therapy. In such a vector, the signal sequence is operably linked to the promoter such that the mRNA transcribed from the promoter encodes the signal peptide. The cloning site is located downstream of the signal sequence so that the gene encoding the protein desired to be secreted can be easily inserted into the vector and fused to the signal sequence. The vector is introduced into a suitable host cell. The protein expressed from the promoter is secreted extracellularly, thereby producing a therapeutic effect. Cellular host Another object of the invention is to transform or transfect with one of the polynucleotides described herein, and in particular a polynucleotide comprising a GENSET regulatory polynucleotide or a polynucleotide encoding a GENSET polypeptide. Host cell. Further included is a host transformed (prokaryotic) or transfected (eukaryotic) with a recombinant vector such as one of those described above.
However, the cell host of the present invention can include any of the polypeptides of the present invention. In a preferred embodiment, the host cell comprises a polynucleotide sequence comprising a sequence selected from the group consisting of the sequences SEQ ID NOs: 1-241, the sequences of clone inserts of the deposited clone pool, variants and fragments thereof. Preferred host cells used as recipients for the expression vector of the invention are: a) Prokaryotic host cells: Escherichia coli strain (IEDH5-α strain), Bacillus subtilis, S. typhi. (Salmonella typhimurium) and Pseudomonas (Pseudomonas), Streptomyces (Streptomyces) and Staphylococcus (Staphylococcus).

B) Eukaryotic host cells: HeLa cells (ATCC No. CCL2; CCL2.1; CCL2.2), Cv 1 cells
(ATCC No. CCL70), COS cells (ATCC No. CRL1650; No.CRL1651), Sf-9 cells (A
TCC No. CRL1711), C127 cells (ATCC No. CRL-1804), 3T3 (ATCC No. CRL-63)
61), CHO (ATCC number CCL-61), human kidney 293. (ATCC number 45504; CRL-1573).
And BHK (ECACC No. 84100501; No. 84111301). c) Other mammalian host cells. The invention also includes primary, secondary, and immobilized homologous recombinant cells of vertebrate origin, preferably of mammalian origin and especially of human origin, which have been engineered as follows: a) exogenous The (heterologous) polynucleotide is the endogenous chromosome of the target gene
Insert into DNA, b) lack endogenous DNA, and / or c) endogenous chromosome
Replace the DNA with an exogenous polynucleotide. Insertions, deletions, and / or substitutions of polynucleotide sequences can be to control regions, such as promoter and enhancer sequences, that are operably associated with the coding sequence of the target gene and / or the target gene. In addition to encompassing host details, including the vector constructs described herein, the invention also includes deletions or substitutions of endogenous genetic material (eg, coding sequences), and / or polynucleotides of the invention. Primary, secondary, of vertebrate origin, particularly mammalian origin, engineered to contain genetic material (eg, a heterologous polynucleotide sequence) operatively associated with and activating, modifying, and / or amplifying an endogenous polynucleotide. Subsequent, and immobilized host cells are also included. For example, methods known in the art can be used to operably associate the endogenous polynucleotide sequence with a heterologous control region (eg, promoter and / or enhancer) by homologous recombination. In this regard, for example, US Pat. No. 5,641,670 issued on June 24, 1997; International patent application publication published on September 26, 1996. WO 96/29411; 1
See International Patent Application Publication WO 94/12650, published Aug. 4, 994; Koller et al. (1989); and Zijlstra et al. (1989). The full text of each publication is incorporated by reference. The present invention further relates to methods of in vitro or in vivo homologous recombinant host cell production in which the expression of the target gene is usually modified. Preferably, the modification results in expression of the target gene under normal growth conditions or conditions suitable for producing the polypeptide encoded by the target gene. The method comprises the following steps: (a) transfecting cells in vitro or in vivo with a polynucleotide construct, said polynucleotide construct comprising: (i) a target sequence; (ii) a regulatory sequence and / or a coding sequence. And (iii) optionally containing an unpaired splice donor site thereby producing a transfected cell; (b) maintaining the transfected cell in vitro or in vivo under conditions suitable for homologous recombination. To do.

The invention further relates to a method of modifying the expression of a target gene in an in vitro or in vivo cell that is not normally expressed, said method comprising the steps of:
(A) transfecting cells with a polynucleotide construct in vitro or in vivo,
The polynucleotide construct comprises (i) a target sequence; (ii) regulatory sequences and / or coding sequences; and (iii) optionally an unpaired splice donor site such that a transfected cell is produced. And (b) maintaining the transfected cells in vitro or in vivo under conditions suitable for homologous recombination thereby producing homologous recombinant cells; and (c) under conditions suitable for gene expression. , Maintaining the homologous recombinant cells in vitro or in vivo. The invention further relates to a method of producing a polypeptide of the invention by modifying the expression of a target endogenous gene in an in vitro or in vivo cell that is not normally expressed, said method comprising the steps of: (a) A cell is transfected with a polynucleotide construct in vitro or in vivo, said polynucleotide construct comprising: (i) a target sequence; (ii) regulatory and / or coding sequences; and (iii).
A transfected cell under conditions suitable for homologous recombination, optionally comprising an unpaired splice donor site thereby producing a transfected cell; and (b) thereby producing a homologous recombinant cell Is maintained in vitro or in vivo;
c) under conditions suitable for gene expression thereby producing a polypeptide,
Maintaining homologous recombinant cells in vitro or in vivo. The invention further relates to polynucleotide constructs that modify the expression of the target gene in cell types in which the target gene is not normally expressed. This occurs when the polynucleotide construct is inserted into the chromosomal DNA of the target cell and said polynucleotide construct comprises: a) target sequence; b) regulatory and / or coding sequences; and c) necessary. Depending on the unpaired splice donor site. The polynucleotide construct further comprises a polynucleotide as described above, wherein said polynucleotide construct further comprises a polynucleotide encoding a polypeptide and is in frame with the target endogenous gene after homologous recombination with chromosomal DNA. The composition is described in US Pat.
54,288; 6,048,729; 6,048,724; 6,048,524; 5,994,127; 5,968,502; 5,965
, 125; 5,869,239; 5,817,789; 5,783,385; 5,733,761; 5,641,670; 5,580,734
Those described by WO 96/29411, WO 94/12650 and Ko;
The method can be carried out as well as produced by techniques known in the art, such as the scientific papers described by Ller et al. (1994). (Each publication is incorporated by reference in its entirety).

The expression of the GENSET gene in mammalian cells, preferably human cells, can be deleted or the endogenous GENSET gene present in the animal's genome can be transformed into GE according to the invention.
It can be modified by substituting with NSET polynucleotides. These genetic modifications can occur by homologous recombination with the particular polynucleotide constructs described above. Mammalian zygotes, such as mouse zygotes, can be used as cell hosts. For example,
1 ng / ml (for BAC inserts) to 3 ng / μl (P1 bacteriophage insert) in 10 mM Tris-HCl, pH 7.4, 250 μM EDTA containing 100 mM sodium chloride, 30 μM spermine and 70 μM spermidine In the case of 1), the mouse zygote undergoes microinjection with the target purified DNA molecule which is the purified DNA molecule prepared in advance. If the DNA to be microinjected is large, Sched
As described by L et al. (1993b), polyamines and high concentrations of salts can be used to avoid mechanical damage to this DNA. The entire text of that disclosure is incorporated herein by reference. One of the polynucleotides of the invention, including the polynucleotide constructs described herein, can be introduced into an embryonic stem cell (ES) line, preferably a mouse ES cell line. ES
The cell line is derived from the inner cell mass of a pluripotent, undifferentiated, preimplantation blastocyst. Preferred ES cell lines are: ES-E14TG2a (ATCC No. CRL-1821), ES-D3 (ATC
C number CRL1934 and CRL-11632), YS001 (ATCC number CRL-11776), 36.5 (ATC
C number CRL-11116). ES cells remain undifferentiated by culturing in the presence of growth-suppressing feeder cells that provide the appropriate signals to preserve this embryonic phenotype and also serve as a matrix for ES cell adhesion. To be done. Preferred feeder cells are primary embryos established from tissue of 13-14 day old embryos of virtually any mouse strain that has been subcultured, as described by Abbondanzo et al. (1993). Fibroblasts, which are growth-suppressed by irradiation as described by Robertson (1987) or under inhibitory concentrations of LIF as described by Pease and Williams (1990). The entire disclosures of these disclosures are incorporated herein by reference. The construct in the host cell can produce the gene product encoded by the recombinant sequence by conventional methods.

After proper transformation of the host and growth of the host to a suitable cell density, the selective promoter is induced by suitable means such as temperature shift or chemical induction and the cells are further cultured for an additional period of time. . Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract stored for further purification. Microbial cells used for expression of proteins can be disrupted by any of the conventional methods including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are known to those of skill in the art. <Transgenic Animal> As used herein, a “transgenic animal” or “host animal” refers to a genome that has been genetically and artificially engineered to contain one of the nucleic acids according to the present invention. The animals that have them are called. A preferred animal is a non-human mammal, Mus, which has a genome artificially and genetically modified by the insertion of a nucleic acid according to the invention.
(Eg: mouse), Rattus (eg: rat), and Oryctogalus (eg: rabbit). In one embodiment, the invention provides a GENS disrupted by homologous recombination with a recombinant vector or knockout vector of the invention.
Includes non-human host mammals and animals that contain the ET gene. All of the transgenic animals of the invention have within their multiple cells recombinant or synthetic DNA sequences, more particularly, or purified or isolated nucleic acids containing GENSET coding sequences as described herein, GENSET regulatory poly. Contains one of the DNA sequences encoding the nucleotide, the polynucleotide construct, the antisense polynucleotide. Generally, the transgenic animal according to the invention comprises a polynucleotide, a recombinant vector and a cell host as described in the invention. In a first preferred embodiment, these transgenic animals, in particular one of the inserted polynucleotides encoding in their genome an unchanged GENSET protein or a mutant GENSET protein.
Alternatively, transgenic animals containing several copies are excellent experimental models for studying the diverse pathologies associated with a given GENSET gene expression ataxia. In a second preferred embodiment, these transgenic animals are capable of expressing the desired polypeptide of interest under the control of the regulatory polynucleotide of the GENSET gene,
This protein of interest is synthesized in high yield, thus leading to tissue-specific expression of the protein of interest.

In a third preferred embodiment, these transgenic animals are capable of expressing the desired polypeptide of interest fused to the GENSET signal peptide sequence, eliciting secretion of the fusion (chimeric) polypeptide. The design of the transgenic animals of the present invention is done by conventional techniques known to those skilled in the art. For further details regarding the production of transgenic animals, particularly transgenic mice, see US Pat. No. 4,873,191 issued Oct. 10, 1989; Nov. 7, 1995.
U.S. Pat. No. 5,464,764 issued on January 4, and U.S. Pat. No. 5,789,21 issued on August 4, 1998.
5, which are incorporated herein by reference for publication of methods for producing transgenic mice. The transgenic animals of the invention are produced by the application of methods that result in animals having a genome that has integrated exogenous genetic material. The method comprises obtaining genetic material that encodes either a GENSET coding sequence, a GENSET control polynucleotide or a DNA sequence encoding a GENSET antisense polynucleotide, or a portion thereof, as described herein. . The recombinant polynucleotide of the present invention is
Inserted into embryo or ES cell line. Insertion is preferably performed using electroporation as described in Thomas et al. (1987), the disclosure of which is incorporated herein by reference in its entirety. Cells undergoing electroporation are searched for positive cells which have incorporated an exogenous recombinant polynucleotide in their genome, preferably via a homologous recombination event (eg by selection marker, PCR or Southern blot analysis). Screened. An exemplary positive-negative selection method that can be used in accordance with the present invention is M
by Ansour et al. (1988), the disclosure of which is incorporated herein by reference in its entirety. Positive cells are then isolated, cloned and injected into 3.5 day old blastocysts from mice as described by Bradley (1987). The entire disclosure of which is incorporated herein by reference. The blastocyst is then inserted into a female host animal and allowed to grow to term. Alternatively, positive ES cells can be prepared as described in Wood et al. (1993) or Nagy et al. (1
993) (the disclosure of which is incorporated herein by reference in its entirety), the ES cells were brought into contact with embryos at the 2.5 day age 8-16 cell stage, Internalize to broadly colonize blastocysts containing cells that give rise to germ cell lines.

[0116] The offspring of the female host may be dependent on which animal is transgenic, eg, insertional exogenous.
It is tested to determine if it contains the DNA sequence and is wild type. The invention therefore also relates to a transgenic animal comprising a nucleic acid according to the invention, a recombinant expression vector or a recombinant host cell. Recombinant Cell Lines Derived from the Transgenic Animals of the Invention A further object of the invention includes recombinant host cells obtained from the transgenic animals described herein. One embodiment of the invention is a recombinant vector of the invention, or a GENS disrupted by homologous recombination with a knockout vector.
Includes cells derived from non-human host mammals and animals that contain the ET gene. Recombinant cell lines are obtained by transfecting a primary cell culture with a vector expressing an onc-gene, such as the SV40 large T antigen, as described by Chou (1989) and Shay et al. (1991), It can be established in vitro from any cell obtained from the tissue of a transgenic animal according to the invention. The entire text of this disclosure is incorporated herein by reference. (Use of Polypeptide of the Present Invention) <Protein Containing Multimerization Domain> The present invention relates to a composition using the protein of the present invention containing a multimerization domain, such as a leucine zipper or a helix loop helix domain, and Regarding the method. The protein of the present invention containing a leucine zipper domain is herein referred to as LZP
And a part thereof, preferably a fragment containing the leucine zipper domain, or a derivative thereof, as described in this section, and containing the leucine zipper as shown in Table VI, Mediates the multimerization of. The leucine zipper consists of periodic repeats of every seventh leucine residue, covering a distance spanning eight helix turns. The segments containing these periodic arrays of leucine residues are thought to be in the alpha-helical conformation, with leucine side chains extending from one alpha helix from a similar alpha helix of the second polypeptide. Interacts with the leucine side chain of erythrocyte to promote dimerization. The structure formed by the cooperation of these two regions forms a coiled coil (O'Shea EK, Rutkowski R., Kim PS Science 243: 538-542., 1989).

Leucine zippers contribute to the targeting of various proteins (eg,
Glucose transporter, Asano et al., J. Biol. Chem., 267, 19636-19641 (1992)).
Furthermore, it enables dimerization of various cytoplasmic hormone receptors and enzymes (Form
an et al., Mol Endocrinol, 3, 1610-1626 (1989)). The leucine zipper also
It is a general feature of protein transcription factors that allow homo or heterodimerization to result in tight binding to DNA strands (see Abel et al., Nature 341, 24-
25 (1989); Jones et al., Cell 61, 9-11 (1990); Lamb et al., Trends in Biochem.
ical Sciences 16, 417-422 (1991)). The leucine zipper has been shown to be a useful tool in several areas of biotechnology, especially protein engineering, with some applications for its ability to mediate homodimerization or heterodimerization. Has been discovered. For example,
Bosslet et al., Describe in particular the use of a pair of leucine zippers for in vitro diagnostics, particularly immunochemical detection and determination of analytes in biological fluids (US Pat. No. 5,643,731) / Tso et al. Leucine zippers for the production of bispecific antibody heterodimers (US Pat. No. 5,932,448) / Soluble oligomeric protein preparation methods using leucine zippers Conrad et al. (US Pat. No. 5,965,712),
Ciardelli et al., (US Pat. No. 5,837,816), Spriggs et al., (WO9410308) / leucine zipper forming sequences have been used by Pelletier et al., In a protein fragment complementation assay to detect biomolecular interactions (WO98).
34120). Due to its utility in biotechnology, the isolation of the new leucine zipper domain is of great interest. Multimerizing activity of proteins containing the leucine zipper domain is described in US Pat.
It can be assayed using any of the assay methods known to those skilled in the art, including circular dichroism spectra described in 2,433 and thermal melting analysis. Alternatively, the leucine zipper motif in LZP can be used by those skilled in the art as a "bait protein" in the wells of the yeast two-hybrid method to generate cDs from different tissues or cell types of a given organism.
It can be used to identify in vivo interacting protein partners from NA libraries. Alternatively, LZP or parts thereof can be used by those skilled in the art of mammalian cell transfection experiments. When fused to an appropriate peptide tag, such as the <His> ₆ tag in a protein expression vector and introduced into cultured cells, this expressed fusion protein can be expressed as a potential intergenic peptide by using an anti-tag peptide antibody. Can be immunoprecipitated with a working protein. This method can be selected for the identification of association partners or confirmation of the results obtained by other methods as described above.

In a preferred embodiment, the present invention comprises techniques known to those skilled in the art, including those described in International Patent Application Publication No. WO9410308, the disclosure of which is incorporated herein by reference in its entirety. A composition and method using LZP or a part thereof for the preparation of a soluble multimeric protein, which is present in a multimer of a fusion protein comprising a leucine zipper fused to a protein of interest. In another preferred embodiment, the LZP or derivative thereof comprises a bispecific antibody heterodimer as described in US Pat. No. 5,932,448, the publication of which is incorporated herein by reference in its entirety. Used to produce. Briefly, leucine zippers capable of forming heterodimers are each bound by an epitope binding member with a different specificity. Bispecific antibodies are formed by the pairwise association of leucine zippers, forming heterodimers that bind two different epitope binding members. In yet another preferred embodiment, LZP or a portion thereof or a derivative thereof is
It is used for the detection and determination of analytes in biological fluids, as described in US Pat. No. 5,643,731, the publication of which is incorporated herein by reference in its entirety. Briefly, the first leucine zipper is immobilized on a solid support and the second leucine zipper is further bound to a specific binding partner of the analyte in biological fluids.
The two peptides are then contacted, thereby immobilizing the binding partner on the solid phase. The biological sample is then contacted with an immobilized binding partner and the amount of analyte in the sample bound to the binding partner is measured. In still other preferred embodiments, LZP or a portion thereof, including, for example, those described in US Pat. No. 5,942,433, the disclosure of which is incorporated herein by reference, inhibits cell growth. And / or can be used to synthesize new nucleic acid binding proteins that can multimerize with the protein of interest for regulation. In another embodiment, the present invention detects in vivo and in vitro biomolecular interactions as described in International Patent Application Publication WO9834120, which publication is incorporated herein by reference in its entirety. For the use of LZP or a portion thereof or a derivative thereof in a protein fragment complementation assay for Such assays can be used to screen cDNA libraries for target proteins that bind unknown proteins or small organic molecules for biological activity, such as protein-protein, protein-nucleic acid, protein-carbohydrate and protein-small molecule. Used to study equilibrium and kinetic aspects of molecular interactions, including interactions.

Still another object of the present invention relates to the use of LZP or a part thereof for identifying a new leucine zipper domain using a protein-protein interaction detection technique known to those skilled in the art. Among the conventional methods available are co-immunoprecipitation,
There are crossover and co-purification methods by cell lysate gradient or chromatographic columns. Once isolated as a protein that interacts with LZP, such an intracellular protein is identified (eg, its amino acid sequence determined) and standard methods are used to identify other proteins with which it interacts. Can be used together. The amino acid sequence thus obtained can be used as a guide for the production of an oligonucleotide mixture which can be used to screen the gene sequences encoding such intracellular proteins. Screening can be accomplished, for example, by standard hybridization or PCR methods. Methods for the production of oligonucleotide mixtures and their screening are known. For example, "Current Protocols in Molecular Biology" edited by Ausubel et al.
, J. Wiley and Sons (New York, NY 1993) and "Guide to PR Protocol Methods and Applications, 1990, edited by Innis, M. et al., Academic Press, Inc., New York." Alternatively, methods known to those of skill in the art can be used to co-identify genes encoding intracellular proteins that can dimerize with LZP or a portion thereof. These methods include, for example, a method in which LZP or a part thereof is labeled in the same manner as a method known as an antibody search method for lambda.gt11 library, or, for example, a marker (for example, enzyme, fluorescence, luminescent protein). , Or pigment) or Ig
-Including a search for a cDNA expression library using a fusion protein such as LZP or a part thereof fused to a Fc domain (see Ausubel et al., Supra for technical details on screening a cDNA expression library). Alternatively, another method of detection of protein in vivo interactions, the two-hybrid method, can be used. [Protein of SEQ ID NO: 261 (Internal Designation Number 116-054-3-0-E6-CS)] The proteins consisting of 233 amino acids of SEQ ID NO: 261 encoded by the cDNA of SEQ ID NO: 20 are 142-163 and 170-191 Two leucine zipper sites are shown in position.

It is believed that the protein of SEQ ID NO: 261 can dimerize by itself (homodimerization) or a heterologous protein of interest (heterodimerization) and via its leucine zipper domain. A preferred polypeptide of the invention is SEQ ID NO: 26.
Polypeptides comprising fragments from positions 142-163 and 170-190 of 1, and fragments having any of the biological activities described herein. [Protein of SEQ ID NO: 263 (Internal Designation No. 116-055-2-0-F7-CS)] The protein of SEQ ID NO: 263 encoded by the cDNA of SEQ ID NO: 22 is NH2
The leucine zipper pattern located near the terminal portion (15-36 positions) is shown. It is believed that the protein of SEQ ID NO: 263 can dimerize by itself (homodimerization) or a heterologous protein of interest (heterodimerization) and its leucine zipper domain. A preferred polypeptide of the invention is SEQ ID NO: 26.
A polypeptide comprising a fragment from positions 15-36 of 3, and a fragment having any of the biological activities described herein. [SEQ ID NO: 245 protein (internal designation number 105-026-1-0-A5-CS)] The protein of SEQ ID NO: 245 encoded by the cDNA of SEQ ID NO: 4 is its COOH
The leucine zipper pattern located near the terminal portion (positions 371 to 392) is shown. It is believed that the protein of SEQ ID NO: 245 can dimerize by itself (homodimerization) or a heterologous protein of interest (heterodimerization) and its leucine zipper domain. A preferred polypeptide of the invention is SEQ ID NO: 24.
A polypeptide comprising a fragment from position 371-392 of 5, and a fragment having any of the biological activities described herein. [Protein of SEQ ID NO: 257 (Internal Designation No. 106-043-4-0-H3-CS)] The 265 amino acid long protein of SEQ ID NO: 257 encoded by the cDNA of SEQ ID NO: 16 is a homosapiens hypothetical protein No .: AJ278482). These two proteins are probably the result of alternative splicing. The protein of SEQ ID NO: 257 shows a leucine zipper located at positions 155-176. It is believed that the protein of SEQ ID NO: 257 may dimerize by itself (homodimerization) or a heterologous protein of interest (heterodimerization) and its leucine zipper domain. Preferred polypeptides of the present invention are polypeptides that include leucine zipper domain fragments and fragments that have any of the biological activities described herein.

[Protein of SEQ ID NO: 314 (Internal Designation No. 188-41-1-0-B8-CS.cor)] An increasing number of proteins are translated by a fatty acid covalently linked to a cysteine residue via a thioester bond. It has been shown to undergo post-modification. Fatty acid modification contributes to intracellular protein localization by promoting membrane binding and further enhancing protein-protein interactions. The cycles of palmitoylation and depalmitoyl have been described for many intracellular proteins, but the relevant enzymes that catalyze these processes have not yet been fully characterized, and the full significance of these cycles is also It remains unclear. Palmitoyl-protein thioesterase-1 (PPT1) is a lysosomal hydrolase that removes long chain fatty acyl groups from modified cysteine residues in proteins. It has been discovered that mutations in PPT1 cause a pediatric form of neuronal ceroid lipofuscinosis (INCL). Soyombo and Hofmann (J. Biol. Chem. 272: 27456-27463 <1997>) showed that PPT2
The cDNA encoding was identified. The putative PPT2 protein contains 302 amino acids, including a leader peptide of 27 amino acids, a sequence motif characteristic of many thioesterases and lipases, and 5 potential N-linked glycosylation sites. P
PT2 shares 18% amino acid identity with PPT1. Soyombo and Hofmann tentatively localized the human PPT2 gene at 6p21.3. Northern blot analysis detected a dominant 2.0-kb PPT2 transcript in the human tissues tested, which had the strongest expression in skeletal muscle and variable amounts of 2.8- and 7.0-kb transcripts were also observed. It was Cellular fractionation studies show the presence of PPT2 in the lysosomal fraction. Immunoblot analysis of recombinant PPT2 expressed in mammalian cells revealed 6 PPT2 proteins ranging in size from 31 to 42 kDa. Asparagine-linked oligosaccharide removal treatment
One major protein, which is kDa, and a minor protein, which is 33 kDa, were produced. Like PPT1, recombinant PPT2 has thioesterase activity and is localized to lysosomes. PPT2 is thought to have a different substrate specificity from PPT1 because PPT2 cannot replace PPT1 in the correction of metabolic defects in INCL cells and thus cannot remove the palmitate group from palmitoylated proteins. However, other studies show that after expression of the recombinant protein in the baculovirus system, using cell lysate as a substrate, this protein has S-thioesterase activity that favors the acyl palmitin and mysteric acid. Was successful.

The present invention provides the protein / polypeptide of SEQ ID NO: 314 encoded by the cDNA of SEQ ID NO: 73. The invention also provides a bioactive fragment of SEQ ID NO: 314. In one embodiment, the polypeptide of SEQ ID NO: 314 is clone 1
88-41-1-0-B8-CS is compatible with the corresponding polypeptide encoded by the human cDNA. A “bioactive fragment” is defined as a peptide or polypeptide that has at least one of the biological functions of a full-length protein (eg,
Removal of long chain fatty acyl groups from modified cysteine residues in proteins). Sequence number
Compositions of 314 proteins / polypeptides or bioactive fragments thereof are also provided by the present invention. These compositions can be made according to methods known in the art. The present invention also provides variants of the protein of SEQ ID NO: 314. These variants have at least about 80% of the amino acids encoded by SEQ ID NO: 73,
Preferably at least about 90%, and most preferably at least about 95% amino acid sequence identity. Variants according to the invention also have at least one functional or structural characteristic of the protein of SEQ ID NO: 314. The present invention also provides bioactive fragments of variant proteins. Compositions of variants, or bioactive fragments thereof, are also provided by the present invention. These compositions can be made according to methods known in the art. Unless otherwise stated, the methods disclosed herein are carried out using the protein encoded by SEQ ID NO: 73, the bioactive fragment of SEQ ID NO: 314, variants of SEQ ID NO: 314 and bioactive fragments of said variants. it can. Due to the redundancy of the genetic code, various DNA sequences can encode the amino acid sequence of SEQ ID NO: 314. In a preferred embodiment, SEQ ID NO: 314 is clone 188-41-1-0-.
It is encoded by B8-CS or the cDNA of SEQ ID NO: 73. It is within the skill of one in the art to create these alternative DNA sequences that encode proteins having identical or substantially identical amino acid sequences. These mutant DNA sequences are therefore within the scope of the invention. As used herein, a "substantially identical" sequence means a sequence having amino acid substitutions, deletions, additions or insertions that does not substantially affect biological activity. Also included in this definition are fragments that retain one or more characteristic biological activities of the protein encoded by clone 188-41-1-0-B8-CS.

In one embodiment of the invention SEQ ID NO: 314 and variants thereof can be used for polyclonal or monoclonal antibody production. The biologically active and immunogenic fragment of SEQ ID NO: 314, or variant proteins can be used for antibody production. Polyclonal and / or monoclonal antibodies can be produced by methods known to those of skill in the art. The antibodies produced in accordance with the present invention can be used in various detection assays known to those of skill in the art. SEQ ID NO: 314 can be used as an identifying marker for lysosomal dysfunction in an individual. In this aspect of the invention, an antibody specific for SEQ ID NO: 314 or a fragment thereof is used in a general immunoassay for screening the presence or absence of SEQ ID NO: 314, or a fragment thereof, in a sample containing lysosomal contents. To be done. Sequence number
The presence or absence of the 314 protein can be used to provide an indication of lysosomal function,
Therefore, it is useful for diagnosis / prognosis identification of lysosomal dysfunction. The invention also provides materials and methods for screening an individual sample for the presence or absence of a nucleic acid encoding the protein of SEQ ID NO: 314, or variants thereof. In one embodiment, the nucleic acids are provided for mRNA or cDNA hybridization assays known to those of skill in the art. Hybridization assay
Performed on nucleic acids obtained or derived from individuals suspected of lysosomal dysfunction. Hybridization assays screen for the presence or absence of nucleic acid encoding SEQ ID NO: 314, or variants thereof. The presence or absence of such nucleic acids can be used as a predictive / prognostic indicator of disease state or lysosomal function. The nucleic acids of the invention can also be used in gene replacement or gene therapy protocols. In this aspect of the invention, the nucleic acid encoding SEQ ID NO: 314 or a bioactive fragment thereof can be introduced into a cell and transplanted into an individual with a lysosomal disorder. In one embodiment, genetically engineered macrophages can be used for therapeutic treatment (eg, Eto and Ohashi <2000> J. Inherit. Metabol.
Dis. 23: 293-298). Alternatively, autologous cells may be obtained from an individual, transformed ex vivo with a nucleic acid, propagated ex vivo and then reintroduced into the individual. Such methods are known to those of skill in the art. [SEQ ID NO: 280 protein (internal designation number 160-75-4-0-A9-CS)] The protein of SEQ ID NO: 280 encoded by the cDNA of SEQ ID NO: 39 and expressed in the fetal brain is located on chromosome 7. Chromosome 12 paralogus of C7orf2, a human protein described as a transmembrane receptor (Heus, HC, A. Hing et al., (1
999) Genomics 57 (3): 342-51). In addition, this protein is an ortholog of the mouse gene LMBR1L that was found to be associated with polydactyly in mice (Clark,
RM, PC Marker, et al. (2000) Genomics 67 (1): 19-27). High levels of homology were also found for the genes identified in Fugu rubripes (AF056116), as well as C. Elegans R05D3.2 (Gellner, K. and S. Brenner (1999) Genome Re.
s 9 (3): 251-8).

The 362 amino acid long protein of SEQ ID NO: 280 encoded by the cDNA of SEQ ID NO: 39 is a splice variant of Z64989 located on chromosome 12.
Chromosome 12 genes are gene bank entries AK001356 and AK001651, GeneS
It has 6 known variants described in eqn entries A26354, A26375, X27360 and Z64989. The closest sequence at the nucleotide or protein level is Z64989. Z64989 splits into 17 exons, of which the protein of the invention comprises the last 14. The transcription start site of the cDNA of SEQ ID NO: 39 is Z64989.
Within the third intron of, the protein of the invention begins at position 128 of Z64989. In addition, two latent leucine zippers are present in the proteins of the invention (positions 136-157 and 272-293). Forelimb polydactyly is a congenital hand malformation that includes double thumbs, various shapes of the phalangeal thumb, and duplication of the index finger. Clark et al. (Supra) demonstrated a correlation between spatial and temporal changes in Lmbr1 expression and development of the embryonic polydactyly phenotype.
It was suggested that the downstream of 1 caused polydactyly. The Lmbr1 gene appears to be involved in limb patterning during mammalian development, for example by the receipt and transduction of localized secretory ligands in the developing limb. The protein of SEQ ID NO: 280 is a paralogus of human C7orf2 and is therefore believed to be involved in the patterning of mammalian body-schedules during early development. For example, the proteins of the invention may be involved in the organization of limb development as well as fetal brain development. As such, the activity of the protein appears to influence various cellular processes including gene expression, cell growth and proliferation, and cell differentiation. In addition, the leucine zipper in the protein allows the protein to undergo specific protein-protein interactions with other leucine zippers, including proteins that include themselves (homodimerization). Preferred polypeptides of the present invention are fragments of SEQ ID NO: 280 that have any of the biological activities described herein. In one embodiment of the invention, the protein can be used to identify fetal brain cells. For example, the proteins of the invention or portions thereof can be used to synthesize specific antibodies using techniques known to those of skill in the art. Such tissue-specific antibodies can then identify tissue of unknown origin, such as differentiated tumor tissue that has metastasized to forensic samples, different body sites, etc., or use immunochemistry to identify different tissue types in tissue cross-sections. Can be used to classify. In addition, labeling reagents capable of specifically binding to the proteins of the invention include cell membrane and intracellular secretory pathway components such as ER.
And Golgi, can be used to observe.

In another embodiment of the invention, the protein is a developmental abnormality or is capable of determining such an abnormality, eg in the fetus or adult (ie is a carrier of a mutated copy of a gene). Can be used to diagnose). An individual found to carry one or two mutated copies of the gene may be, for example, a gene therapy or other strategy for correcting or compensating for a genetic defect, or that the child is not a carrier of the mutated gene. Would be a candidate for a strategy to confirm. Characterization of mutations in the gene encoding this protein is also of great value in understanding the nature of polydactyly and other developmental disorders, and thereby in other strategies for the treatment and prevention of these disorders. Promote development. In other embodiments, the protein is used to modulate gene expression, cell growth and proliferation, and / or cell differentiation in cells in vitro or in vivo. For example, any of these behaviors may increase or suppress cell growth in vitro, such as, for example, protein production or ex vivo therapeutic strategies. Moreover, any of these diseases associated with increased or decreased in vivo cellular behavior can be treated or prevented by enhancing or suppressing the expression or activity of the proteins of the invention in in vivo cells. [Proteins of SEQ ID NOs: 309 and 304 (Internal Designation Numbers 188-11-1-0-B3-CS and 187-34-0-0-l12-CS)] SEQ ID NOs: 309 and 304 are SEQ ID NOs: 68 and It is encoded by 63 cDNAs. Therefore, all the features and uses of the polypeptides of SEQ ID NOs: 309 and 304 described throughout this application also apply to clones 188-11-1-0-B3-CS and 187-34.
It will be appreciated that it relates to the polypeptide encoded by the human cDNA which is -0-0-112-CS. In addition, all features and uses of the nucleic acids of SEQ ID NOs: 68 and 63 described throughout this application are also found in clones 188-11-1-0-B3-CS and 187-34-0.
It will be appreciated that it relates to the nucleic acid encoded by the human cDNA which is -0-112-CS. The protein of SEQ ID NO: 309 (encoded by the clone having internal designation number 188-11-1-0-B3-CS) and SEQ ID NO: 304 (clone having internal designation number 187-34-0-0-l12-CS) The polymorphic variant, which is encoded by, and differs in one amino acid from the polypeptide encoded by the clone having the internal designation number 188-11-1-0-B3-CS, is LGI1 (leucine-rich gene- Glioma inactivation)
Up to the first 279 amino acids of the protein are highly homologous. Clone 188-11-
1-0-B3-CS and 187-34-0-0-l12-CS appear to be splicing and polymorphic variants of LGI1. The LGI1 protein is 557 amino acids in length. (Somerville et al. (2000) Mammalian Genome 11, 622-627; Chernova et al. (1998) Oncogene 1
See 7, 2873-2881. Each disclosure is incorporated herein by reference in its entirety). Clone 188-11-1-0-B3-CS aligns with the first 279 amino acids of LGI1 and has 12 amino acids (VLREIHRF) that are not considered homologous to LGI1.
TNMS) is added to the C-terminus. Like LGI1, clone 188-11-1-0-B3-CS and polymorphic variant 187-34-0-0-l12-CS contain the LRP domain and are strongly expressed in brain tissue.

LGI1 belongs to a large family of leucine rich repeat (LRP) proteins. The LRR domain is believed to act as a protein-protein interaction region. It is materialized when a family of known LRR proteins grows. Leucine-rich repeats are essential components in glycoprotein hormone receptors, proteoglycans, and Trk proteins,
It has been identified by biochemical analysis of the expression of mutants and artificial chimeras in tissue culture and the properties of these constructs. Many transmembrane LRR proteins are known or suspected to encode truncated forms of functional significance (eg N and L ⁶ , and Slit). The secreted protein proteoglycan decorin binds TGF-β, a growth factor that stimulates decorin expression. Decorin suppresses the growth of cultured cells and may therefore form part of a negative feedback loop to modulate cell growth. This is similar to the function proposed for the LGI1 receptor protein. Analysis of cerebral glioma indicates that LGI1 expression is abolished or significantly reduced in high-grade tumors compared to more benign tumors, pointing to its role as a tumor suppressor gene (Cowell et al., 2000; Cowell et al., 1998, the entire disclosure of which is incorporated herein by reference). Most glioblastoma multiforme (GBM) brain tumors contain only one gene copy of LGI1, so it is rarely expressed. It is not clear how genes are inactivated, but one possibility is that chromosomal or gene rearrangements that occur in 20-25% of tumors result in inactivation as a result of position effects. It is to be. Recently, 10 LGI1 genes
It was located at q24, was disrupted by translocations in the T98G GBM cell line, and was also determined to be rearranged in 26% abnormalities of primary brain tumors. Alternatively, LGI1
Can be part of a highly regulated pathway, where inactivation of other key members or highly specific transcription factors results in either inactivation of all genes in the pathway or failure of transcription initiation. Since functional inactivation of LGI1 occurs during the transition from low-grade to high-grade brain tumors, knockout or transgenic mice with reduced, eliminated or modified expression of the protein of SEQ ID NO: 309 or 304 can be used as a disease model . In particular, mice overexpressing LGI1 can be used as a tumor development model.

Since human and mouse LGI1 are highly conserved, showing 91% identity at the nucleotide level, 97% similarity at the amino acid level, and most amino acid substitutions being conserved, mice are It is particularly useful as a model for assessing the consequences of protein level or activity modifications of SEQ ID NO: 309 or 304, as well as for identifying agents effective in the treatment of tumorigenesis. The mouse lgi1 gene is 4.2 kb long, but human LGI1
1 is 2.2 kb. The size difference between the human and mouse genes is the result of inclusion of a 2 kb sequence in the 3'untranslated region of the mouse gene. It is not clear whether the additional sequences affect gene expression. Further analysis of the genomic sequence also reveals that the number of exon / intron boundaries is also similar in humans and mice. The high degree of LGI1 conservation between mouse and human implies that this gene is undergoing strong selection forcing. It is interesting to speculate that large biases in the primary protein sequence may cause loss of function of this gene product.
Total or partial loss of LGI1 gene function is therefore lethal, which is
It means that LGI1 plays an important role in normal brain development as well as tumor formation. SEQ ID NOs: 309 and 304 also have high homology to Slit, a Drosophila secretory protein that plays a role in central nervous system axon pathway development. The Slit protein is required for the normal development of the midline in the CNS, especially the midline glial cells, as well as the simultaneous formation of commissural axon pathways. This process depends on Slit protein expression levels. The Slit protein appears to be excreted by median glial cells, where it is its synthetic site and eventually associates with the surface axons that traverse it. Contacting cells with the supernatant expressing this gene product increases the permeability of THP-1 monocytes to calcium. Thus, the Slit protein appears to be involved in the signaling pathway initiated when it binds to its receptor on the surface of monocytes. Because of the above, the proteins of SEQ ID NOs: 309 and 304 are believed to be involved in receptor-mediated signal transduction pathways that modulate cell differentiation and / or proliferation. Analysis by Northern blot detects LGI1 transcripts only in brain, nervous tissue and skeletal muscle, but not in heart, lung, placenta, liver, or pancreas. Northern blot analysis of RNA from several different regions of the human brain showed widespread LGI1 expression, albeit with different intensities. The most abundant were the cerebral cortex, hippocampus, and putamen. The lowest expression was detected in the corpus callosum. Expression levels were moderate in other brain regions. Thus, the protein of SEQ ID NO: 309 or 304 or a fragment thereof, and the polynucleotide encoding the protein of SEQ ID NO: 309 or 304 may be derived from a tissue sample of brain origin (and particularly cerebral cortex, hippocampus, or putamen), neural tissue, and It can be used to determine if it is from skeletal tissue and to identify whether the tissue sample is from the brain or other tissue such as heart, kidney, lung, placenta, or pancreas.

Accordingly, the present invention provides at least 5, 8, 10, 12, 15, 20, 20, which has the desired biological activity in an individual to treat or ameliorate the conditions listed above.
25, 30, 35, 40, 50, 60, 75, 100, 150 or 200 contiguous amino acids,
Or including the use of the protein or fragment of SEQ ID NO: 309 or 304, including fragments. In such embodiments, the protein of SEQ ID NO: 309 or 304, or a fragment thereof, is tumor arrest, modulation of neurogenesis or intervention in brain tumors, glioblastoma multiforme, brain trauma, neurodegenerative disease states and Alzheimer's disease. , Recurrent processes in behavioral disorders such as Parkinson's disease, Tencan, multiple sclerosis, Huntington's chorea, schizophrenia, obsessive-compulsive disorder, and spinal cord injury, cerebral hemorrhage, facial nerve injury, nerve injury resulting from diabetes, and It is administered in an individual where it is desired to increase or decrease any of the protein activities of SEQ ID NO: 309 or 304, including retinal regeneration. The protein of SEQ ID NO: 309 or 304 or fragment thereof may be administered directly to the individual, or the nucleic acid encoding the protein of SEQ ID NO: 309 or 304 or fragment thereof may be administered to the individual. Alternatively, SEQ ID NO: 309 or 30
An agent that increases the protein activity of 4 may be administered to the individual. Such an agent comprises contacting a protein or a cell of SEQ ID NO: 309 or 304 or a preparation containing the protein of SEQ ID NO: 309 or 304 with a test drug to analyze whether the test drug increases protein activity. Can be identified by For example, the test agent can be a compound or polypeptide or peptide. Alternatively, the protein activity of SEQ ID NO: 309 or 304 can be reduced by administering to the individual an agent that interferes with such activity. An agent that interferes with the activity of the protein of SEQ ID NO: 309 or 304 contacts the test agent with the protein or cell or or a preparation containing the protein of SEQ ID NO: 309 or 304, and the test agent reduces the protein activity. It can be identified by analyzing whether or not. For example,
The test agent can be a compound, polypeptide or peptide, antibody, or nucleic acid such as an antisense nucleic acid or a triple helix forming nucleic acid. In one embodiment, the invention provides for selectively identifying a tissue, preferably the brain, based on the protein level of SEQ ID NO: 309 or 304 in a sample, or
Methods and compositions using the proteins of the invention or portions thereof as marker proteins for identifying the source from which one or more tissue samples may be derived. For example, the protein of SEQ ID NO: 309 or 304 or fragments thereof can be used to raise antibodies using techniques known to those of skill in the art, including those described herein. Such tissue-specific antibodies can then identify tissue of unknown origin, such as differentiated tumor tissue that has metastasized to forensic samples, different body sites, etc., or use immunochemistry to identify different tissue types in tissue cross-sections. Can be used to classify. In such methods, a tissue sample is contacted with a detectably labeled antibody under conditions that promote antibody binding. The level of antibody binding to the test sample is measured and compared to the level of binding to control cells from brain or non-brain tissue to determine if the test sample is brain derived. Alternatively, the protein level of SEQ ID NO: 309 or 304 in the test sample may be measured by determining the RNA level encoding the protein of SEQ ID NO: 309 or 304 in the test sample. RNA levels can be measured using nucleic acid arrays or by methods such as in situ hybridization, Northern blot, dot blot or other methods known to those of skill in the art. The RNA level in the test sample is compared to that of control cells from brain or non-brain tissue to determine if the test sample is from brain. For many disorders listed above, particularly the nervous system, encodes a polypeptide of SEQ ID NO: 309 or 304, as compared to standard gene expression levels (ie, expression levels in healthy tissue or fluid from individuals not suffering from the disorder). Significantly high or low levels of gene expression can be associated with specific tissues or cell types (eg cancerous and injured tissues) or body fluids (eg semen, plasma, synovial fluid and spinal fluid) or such disorders. This can be detected by conventional means in the tissues of other cell samples taken from individuals who have it.

In another embodiment, an antibody to the protein of SEQ ID NO: 309 or 304 or a portion thereof is used to detect cells expressing the protein of SEQ ID NO: 309 or 304 using methods including methods known to those of skill in the art. It can be used for concentration or purification. For example,
Antibodies to the proteins of SEQ ID NO: 309 or 304 or fragments thereof can be immobilized on a solid support such as a chromatography matrix. The preparation comprising cells expressing the protein of SEQ ID NO: 309 or 304 is arranged to contact the antibody under conditions that promote binding to the antibody. The cells are released from the support by washing the support and then contacting the support with an agent that dissociates the cells from the antibody. In another embodiment of the invention, the protein of SEQ ID NO: 309 or 304 or a fragment thereof can be used to diagnose disorders associated with modified expression of the protein of SEQ ID NO: 309 or 304. In certain embodiments, the protein of SEQ ID NO: 309 or 304 or a fragment thereof can be used for cancer diagnosis. In such methods, the protein level of SEQ ID NO: 309 or 304 in a diseased individual is measured using methods such as those described herein and compared to the level in a healthy individual. For example, reduced protein levels of SEQ ID NO: 309 or 304 compared to a healthy individual suggests that the sick individual may have cancer, or may be predictively diagnosed to have cancer in the future. Another embodiment of the invention is a polypeptide comprising the structural or functional domain of the protein of SEQ ID NO: 309 or 304. Such a structural or functional domain of the protein of SEQ ID NO: 309 or 304 is located at a leucine rich repeat C-terminal domain located at amino acid positions 173-222, a leucine rich repeat located at amino acid positions 92-115, at amino acid positions 116-139. A signal comprising the leucine rich repeat located, the leucine rich repeats located at amino acid positions 140-163, the leucine rich repeat located at amino acid positions 164-185, the membrane span segment located at amino acid positions 15-35, and the sequence FLCLLSALLLTEG / KK. Contains peptides. Thus, the polynucleotide of SEQ ID NO: 309 or 304 or a fragment thereof, or a polynucleotide encoding those proteins or fragments thereof, is:
It can be used in an in vitro diagnostic assay for malignant brain tumors such as glioblastoma multiforme. These proteins or nucleic acids can also be found in behavioral disorders such as brain tumors and / or brain trauma, neurodegenerative disease states and Alzheimer's disease, Parkinson's disease, Tencan, multiple sclerosis, Huntington's chorea, schizophrenia, obsessive-compulsive disorder, And can be used to attenuate / prevent / and / or treat spinal cord injury, cerebral hemorrhage, facial nerve injury, nerve regeneration process in nerve injury due to diabetes, and retinal regeneration.

In addition, the proteins, antibodies directed against the proteins, and related small molecules can be used as tumor markers and / or immunotherapeutic targets for the above disease states. For example, because of the homology of the proteins of SEQ ID NOs: 309 and 304 to the secreted form of LGI1, antibodies directed against the amino acids VLREIHRFTNMS of both clones assist in the differential detection of secretory and receptor forms of the protein. obtain. In addition, the proteins of SEQ ID NOs: 309 and 304 or fragments thereof can be used to identify binding partners as described herein. <DNA binding protein> The present invention includes the protein of the present invention containing a DNA binding domain referred to as DBP such as those described in this section, and those containing a DNA binding domain as shown in Table VI, or a part thereof. , Compositions and methods using a fragment, preferably comprising a DNA binding domain, or a derivative thereof. Transcriptional control is primarily achieved by sequence-specific binding of proteins to DNA and RNA. Among known protein motifs related to sequence-specific recognition of DNA, Zn finger proteins are unique in their modularity. Zn finger domains are found in many zinc-binding proteins involved in protein-nucleic acid interactions. They are individually folded zinc-containing minidomains that are used in a modular repeat fashion to achieve sequence-specific recognition of DNA (Kl
ug 1993 Gene 135, 83-92). Such zinc-binding proteins are generally associated with gene expression and usually act as transcription factors (US Pat. No. 5,866,325; 6,
013,453 and 5,861,495). To date, Zn finger proteins containing 2-37 modules have been identified. Over 200 proteins, many of which are transcription factors, have been shown to have Zn finger domains. Zn fingers primarily connect transcription factors to their target genes by binding to specific sequences in DNA. Zn finger modules are found in a wide range of eukaryotic transcriptional regulatory proteins. Zn finger domains generally consist of 25-30 amino acid residues that form one or more tetrahedral ion binding sites. The binding site comprises a ligand consisting of cysteine, histidine and sometimes an aspartate or glutamate side chain. Zinc binding allows relatively short-range polypeptides to fold into defined structural units suitable for participating in macromolecular interactions (
Berg, JM et al. (1996) Science 271: 1081-1085). The Zn finger domain was first recognized in the transcription factor TFfIIIA from the frog (Xenopus) oocyte (Mille
r et al., EMBO, 4: 1609-1614, 1985; Brown et al., FEBS Lett., 186: 271-274, (1
985)).

The Zn finger binding domain containing the C ₃ HC ₄ sequence motif is known as the RING domain (Lovering, R. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 2112-.
2116). The RING domain consists of a sequence that binds eight metal binding residues and two metal ions in an overlapping manner (Barlow, PN et al. (1994) J. Mol. Biol. 237: 201-21.
1). The functions of RING finger proteins are mediated by DNA binding and include gene expression, DNA recombination, and DNA repair (Borden and Freemont, Curr Opin Struct Biol.
6: 395-401 (1996) and US Pat. No. 5,861,495). Both RING fingers and LIM domains are involved in transcriptional regulation, either by mediating protein-protein interactions and directly affecting transcription or by recruiting coactivators or co-repressors. The LIM domain also contributes to various signaling pathways. They can interact with protein kinases to anchor gene products to large protein complexes or cellular compartments. PHD finger is a C ₄ HC ₃ Zn-finger ranging from about 50 to 80 residues, RIN
Distinguished from G-fingers or LIM domains. They are mostly DNA or RNA
It is thought to be a binding domain, but may also be involved in protein-protein interactions (see Aasland et al., Trends Biochem Sci 20: 56-59 for details).
(1995)). The PHD finger domain, which belongs to the Zn finger domain family, is found in numerous regulatory proteins often associated with chromatin-mediated transcriptional regulation. Nucleic acid binding activity of DBP or portions thereof can be assayed using any of the assays known to those of skill in the art, including those described in US Pat. No. 6,013,453. The present invention relates to compositions and methods for gene transcription stimulation using DBP or a part thereof, in particular a fragment containing a DNA binding domain. One of the surprising features of the activation domain of common transcription factors is that when "fusing" it to a heterologous protein domain, is recruited to a wide range of promoters,
That is, the ability to activate transcription is hardly affected. Due to the high functional independence of these activation domains, gene expression, protein-protein, protein-
It will be a valuable tool in various biological assays for analyzing RNA, or protein-small molecule drug interactions. Several strategies have been reported for improving the potency of activation domains, as well as improving gene expression under the control of activation domains. These methods generally involve increasing the copy number of the activation domain fused to the DNA binding domain, or the production of activators, including a synergistic combination of activation domains.

Accordingly, in another embodiment, the invention provides compositions and methods that include a novel transcription factor that comprises a DBP or a portion thereof, preferably a fragment that includes a DNA binding domain. Such transcription factors can be designed to modulate the expression of the target gene of interest. Embodiments of the invention are applicable to systems that include covalent or non-covalent binding of the transcription activation domain to the DNA binding domain. In practice, ie, at least two mutually heterologous domains, one of which is a DNA binding domain of the invention, and in some cases, additional nucleic acid constructs to allow ligand-dependent regulation of target gene transcription. The cells can be engineered by the introduction of a recombinant nucleic acid encoding a fusion protein, including. Administration of the ligand to the cells is then
Modulates target gene transcription (positively, or in some cases negatively) (all experimental methods for this embodiment are fully described in US Pat. No. 6.015.709, the disclosure of which is fully incorporated herein. Incorporated as a reference). Examples (non-limiting) of heterologous domains that may be included with the DNA binding domain in the various fusion proteins of the invention include other transcription control domains (ie, transcription activation domains such as p65, VP16 or AP domains); Enhancing or synergistic domain, or ssn
-6 / TUP-1 domain or transcriptional repression domain such as Kuppel family suppressor domain); DNA binding domain such as GAL4, lex A or complex DNA complex DNA binding domain such as Zn finger domain or ZFHD1 domain; or (
a) an immunophilin, cyclophilin or FRB domain; (b) an antimicrobial binding domain such as tetR or (c) a ligand binding domain containing or derived from a hormone receptor such as a progesterone receptor or an ecdysone receptor . Although a wide range of ligand binding domains can be used in the present invention, ligand binding domains that bind cell penetrating ligands are preferred. It is also preferred that the ligand has a molecular weight of about 5 kD or less, more preferably 2.5 kD or less, optimally about 1500 D. Even more preferred are non-proteinaceous ligands. Examples of ligand binding domains / ligand pairs that can be used in the practice of the present invention are FKBP: FK1012, FKBP: synthetic divalent FKBP ligand (W
O 96/0609 and WO 97/31898), FRB: rapamycin / FKBP (eg W
O96 / 41865 and Rivera et al., "A humanized system for pharmacologic control of gene expressio".
n ") Nature Medicine 2 (9): 1028-1032 (1997)), cyclophilin: cyclosporine (see, for example, WO 94/18317), DHFR: methotrexate (eg, Licitra et al., 1996, Proc. Natl. Acad. Sci. USA 93: 12817-12821), TetR: tetracycline or doxycycline or other analogues or mimics thereof (Gossen and Bujard, 1992, Proc. Natl. Acad. Sci. USA.
89: 5547; Gossen et al., 1995, Science 268: 1766-1769; Kistner et al., 1996,
Proc. Natl. Acad. Sci. USA 93: 10933-10938), Progesterone receptor: RU486 (Wang et al., 1994, Proc. Natl. Acad. Sci. USA 91: 8180-81).
84), ecdysone receptor: ecdysone or muristerone A or other analogs or mimetics thereof (No et al., 1996, Proc. Natl. Acad. Sci. USA 93: 3346-).
3351) and DNA gyrase: coumermycin (eg, Farrar et al., 1996, N.
ature 383: 178-181). In many applications it is preferable to use a DNA binding domain that is heterologous to the cells to be engineered. In the case of complex DNA binding domains, constituent peptide moieties that are endogenous to the cell or organism undergoing the engineering treatment are generally preferred.

In another aspect of this embodiment, the polynucleotides encoding the DNA binding domain as well as other functional fragments of DBP, such as those modulated by tetracycline, RU486 or ecdysone, or an analogue or mimetic thereof. Allostery-based system, and FK1012, FKCsA, rapamycin,
Fusion proteins of various regulated gene expression systems, including both dimerization-based systems such as those modulated by divalent compounds such as AP1510 or coumermycin, or analogs or mimetics thereof. It may be incorporated into the encoding polynucleotide. All this is explained below (also Clacks
on, "Controlling mammalian gene expression by small molecules"
xpression with small molecules), Current Opinion in Chem. Biol. 1: 210-
218 (1997)). The fusion protein has a bundling dom
ains), a DNA binding domain, a transcriptional activation (or repression) domain and a ligand binding domain, which may include a combination of related components. Other heterologous domains may be included. Another embodiment of the invention relates to an expression system, preferably a vector and vector-containing cells, using DBP or a part thereof, in particular a DNA binding domain. In this regard, a fusion protein comprising a transcriptional activation domain of the invention and at least another domain heterologous thereto is encoded, the peptide sequence of said activation domain itself being compared to its counterpart comprising an unchanged peptide sequence. In order to increase or decrease its potency as a transcriptional activator, recombinant nucleic acids are eventually provided which are modified when compared to the naturally occurring sequences from which they were derived. Each of the recombinant nucleic acids of the invention further comprises an expression control sequence operably linked to the coding sequence and can be provided in a DNA vector, eg, for use in prokaryotic or eukaryotic cells. As explained above, some of the recombinant nucleic acids of a given composition, including optimal recombinant nucleic acids, include:
It may be present in one vector or may be distributed between two or more vectors. Recombinant nucleic acids can be provided as inserts in one or more recombinant viruses that can be used to transduce, for example, in vitro cells or cells present in organisms including human or non-human mammalian subjects. To deliver the recombinant nucleic acids of the invention to cells within a recipient organism, non-viral methods (naked DNA,
It should be understood that liposomes or other lipid compositions) can be used. Produced engineered cells and their progeny that contain these recombinant nucleic acids or nucleic acid compositions of the present invention can be used in human gene therapy, similar veterinary applications, or in the production of cells or animal models, including transgenic applications. And in a variety of important applications including assay applications. Such cells are useful, for example, in methods involving the addition of a ligand to the cell, preferably a cell-permeable ligand (or administration of the ligand to an organism containing the cell), for the control of expression of a target gene.

The present invention also relates to methods and compositions that use DBPs or portions thereof to bind nucleic acids, preferably alone or in combination with other materials, DNA. For example, DBP or a portion thereof is added to a sample containing nucleic acid under conditions that allow it to bind to and bind to nucleic acid. In a preferred embodiment, DBP or portions thereof can be used to purify nucleic acids such as restriction fragments. In other preferred embodiments, DBPs or portions thereof can be used to visualize nucleic acids as the polypeptide binds to the appropriate fusion partner or as detected by antibody search. Alternatively, DBP or a portion thereof, alone using techniques known in the art to create affinity chromatography columns,
Alternatively, it may be combined with other DNA binding proteins and bound to a chromatographic support. The sample containing the nucleic acid to be purified is passed through the column. DB on support
Immobilizing P or a portion thereof is particularly advantageous for those embodiments in which the method is practiced on a commercial scale. This immobilization facilitates removal of the protein from the batch product and subsequent reuse of the protein. Immobilization of DBP or a portion thereof can be achieved, for example, by inserting a cellulose binding domain into the protein. It will be appreciated by those skilled in the art that other methods of immobilization can also be used and are described in the published literature. In another embodiment, the invention relates to compositions and methods that use DBPs or portions thereof, particularly DNA binding domains, to modify gene expression of interest in target cells. Such genes of interest are described in US Patents 5,861,495; 5,866,325 and 6,01
It may be a disease-associated gene such as an oncogene, or an exogenous gene from a pathogen such as a bacterium or virus, known to those of skill in the art (using methods), including those described in 3,453. In yet other embodiments, the DBP or portion thereof is cancer and hyperaldosterone, hypoadrenocorticism (Addison's disease), hyperthyroidism (Graves' disease), hypothyroidism, colorectal polyps, Diagnosis, treatment and / or disorders associated with dysfunction of gene transcription such as gastritis, gastric and duodenal ulcers, ulcerative colitis, and other disorders associated with abnormal cell differentiation, proliferation or degeneration, including Crohn's disease
Or it can be used for prevention.

[Protein of SEQ ID NO: 388 (Internal Designation Number 109-002-4-0-C6-CS)] The protein of SEQ ID NO: 388 encoded by the cDNA of SEQ ID NO: 147 has a Zn finger domain, mainly 329 to 339. 375 with PHD finger domain from position
It is a protein having a length of individual amino acids. PHD fingers were first identified by comparing the maize homeodomain (HD) protein ZMHOX1a with the Arabidopsis related HAT3.1 (Bellmann R. et al.
Werr W. EMBO J. 11: 3367-3374 (1992)), named the plant homeodomain (PHD) finger for association with DNA-bound HD in both genes. This motif is trith
orax (TRX-G) or polycomb (PC-G) group (Aasland R. et al., Trends Biochem.Sc
i. 20: 56-59 (1995)), and leukemia-related proteins (LAP fingers) (Saha
V. et al., Proc. Natl. Acad. Sci. USA 92: 9737-9741 (1995)), appearing in various regulatory genes. The established function of the TRX-G and PC-G genes in the Drosophila chromatin modifying action has led to the suggestion that PHD fingers are involved in chromatin-mediated transcriptional regulation. According to recent data, PHD
Are Finger Proteins Associated with the Chromatin Remodeling Complex (Bochar DA
Et al., Proc. Natl. Acad. Sci. USA 97: 1038-1043 (2000)) or contributing to histone acetylation (Loewith R. et al., Mol. Cell. Biol. 20: 3807-3816 (20).
00)) has been proven. Based on the unique His residue position, the PHD finger (Cys4-Hi
The cysteine skeleton of (s-Cys3) is distinctly different from the RING finger (Cys3-His-Cys4) and the LIM domain (Cys2-His-Cys5), and the DRIL domain in which two RING finger motifs are closely linked. In contrast to the large amount of information reported on LIM domains, little functional data on PHD fingers remains (for details, see Halbach T. et al., Nucleic Acids Research 28).
: 3542-3550 (2000)). GYMNOS, a recently described member of the SWI2 / SNF2 protein family (22) in plants, also contains PHD fingers and plays a role in developmental regulation. The second PHD motif of the Drosophila dMI-2 protein (reference for animal counterparts) shares a high degree of sequence conservation with known plant PHD fingers. Due to its similarity to the Drosophila MI-2 gene, GYMNOS is involved in chromatin modifying activity. Although the PHD finger is an isolated motif in GYMNOS, the characteristic Cys4-His-Cys3 skeleton in the PHDf-HD plant gene is embedded in a large region.
This region shares 60% identical residues between 7 genes from different plant families and is more highly conserved than HD (40%). This conservation suggests that PHD fingers are part of a large functional unit. When associated with the leucine zipper in the conserved 180 amino acid region surrounding the PHDf-HD protein, PHD finger activity is masked and silenced. The leucine zipper upstream of the PHD finger mediates the interaction of the plant 14-3-3 protein with the helix 4 and thus identifies the PHDf-HD protein as a potential target of the 14-3-3 signaling pathway. The 14-3-3 family of multifunctional proteins is highly conserved in animals, plants and yeast. Because of the ability of 14-3-3 proteins to form dimers and homo- and heterodimers, 14-3-3 protein family members function as scaffolds that promote protein complex association. 14-3
-3 proteins include various proteins including Raf, BAD, Bcr / Bcr-Abl, KSR (kinase inhibitor of Ras), PKC, PI-3 kinase (phosphorase) and cdc25C phosphatase (dephosphorylate). Involved in signal pathways. Others enter the core,
It is associated with the DNA binding complex. Recent data show contact with TBP, TFIIB and the human TBP-related factor hTAF (II) 32 (see Halsbach T. (supra) for details).

PHD fingers have recently been shown to activate transcription in yeast, plant and animal cells. Transcriptional activation in animal cells tested with different PHD fingers (zebrafish embryos as the test system) resulted in PH in eukaryotic cells.
It appears to be a general feature of the D-finger motif. Whether the PHD fingers interact directly with components of the transcription initiation complex or whether their positive effects on transcription are mediated through accessory protein interactions remains to be elucidated. However, both hypotheses involve PHD finger-mediated protein-protein interactions. The surrounding sequence sterically hinders access to PHD fingers, whose exposure ultimately depends on the binding of protein partners. PHD fingers containing proteins appear to be associated with human disease. Because all clinically significant mutations in the AIRE gene match modifications in two PHD fingers, resulting in the rare autoimmune polyglandular endocrine disorder-Candidiasis-ectodermal dystrophy (APECED) For research on the human AIRE gene (Naga
mine K. et al., Nat. Genet. 17: 393-398 (1997), Scott HS et al., Mol. Endocrin.
ol. 12: 1112-1119 (1998)) clarified the importance of this motif. Presence of PHD fingers in genes up-regulated in leukemia, such as TRX-G or PC-G genes, associated with autoimmune disease APECED or involved in the modification of true chromatin to heterochromatin Indicate that this motif may be involved in a variety of important cellular events including developmental disorders, tumors and immune disorders. For example, the role of remodeled chromatin structures in cancer metastasis and tissue carcinogenesis has been well documented (Zhang Y. et al. Cell 16: 279-289 (19).
98); Klugbauer S. and Rabes HM Oncogene 29: 4388-4393 (1999)). It is believed that SEQ ID NO: 388 or a protein thereof is a zinc binding protein, preferably capable of binding a nucleic acid, more preferably a transcription factor. A preferred polypeptide of the invention is a polypeptide comprising the amino acids of SEQ ID NO: 388 from positions 329-339. Other preferred polypeptides of the present invention are fragments of SEQ ID NO: 388 having any of the biological activities described herein. In one embodiment of the invention, the protein of the invention, or a portion thereof, or a derivative thereof, is a developmental disorder and / or cell associated with dysregulation of gene expression mediated by the PHD finger domain of the protein of the invention. It can be used as a target for the diagnosis of proliferative disorders. Such frost damage may include renal tubular acidosis (acidosis), anemia, Cushing's syndrome, dwarfing cartilage dwarfism, tencan, gonadal dysplasia, Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism. , Hereditary neuropathy such as hydrocephalus, seizure disorders such as Sydenham's chorea and cerebral palsy, bifidular and congenital glaucoma, cataract, sensorineural hearing loss, benign tumor, and adenocarcinoma, leukemia; malignant black Tumors; lymphomas; cancers such as sarcomas; and including but not limited to bladder cancer, colon cancer, liver cancer, brain cancer, small bowel cancer, colon cancer, breast cancer, ovarian cancer, kidney cancer, lung cancer and prostate cancer . Diagnosis may be performed using methods known to those of skill in the art, including Northern blots, RT-PCR, immunoblotting, immunochemistry, enzyme linked immunosorbent assay (ELISA) as described herein. Can be performed using a nucleic acid or antibody that can detect Target sample,
The amount of protein of the invention expressed in the disease derived from biopsied tissues or body fluids or cell extracts taken from controls and patients is compared to standard values. Deviation between standard and control values establishes the parameters of disease diagnosis.

In another embodiment, an antagonist or inhibitor of the protein of the invention or a portion thereof may be administered to a patient for the treatment and / or prevention of the disorders described above. Transcriptional activator antagonists or inhibitors can actually be used to suppress transcriptional activation in tumor cells. Such antagonists and / or inhibitors are specific for the proteins of the invention, which can be used directly as antagonists or indirectly as a targeting or delivery mechanism for bringing the drug to cells or tissues expressing the proteins of the invention. Antibody. Neutralizing antibodies (ie, those that inhibit protein-protein interactions) are particularly preferred for therapeutic use. Other methods for inhibiting the expression of the proteins of the invention include antisense and triple helix strategies as described herein. Other antagonists or inhibitors of the proteins of the invention may be produced using methods known in the art, including screening drug libraries to identify those that specifically bind the proteins of the invention. The proteins of the invention, parts thereof, preferably functional or immunogenic fragments thereof, or oligopeptides related thereto, can be used for screening compound libraries in various drug screening techniques. The fragments used in such screens may be free in solution, attached to a solid support, cell surface, or intercellularly located. The formation of a binding complex between the protein of the invention, or a part thereof or a derivative thereof and the agent to be tested can be determined. Other techniques for drug screening that can be used include high throughput screening of compounds with suitable binding affinity for the proteins of the invention, as described in published International Patent Application Publication WO 84/03564. Supply. [Protein of SEQ ID NO: 394 (internal designation number 157-17-2-0-C1-CS)] The protein of SEQ ID NO: 394 encoded by the extended cDNA of SEQ ID NO: 153 is a myc form from amino acid positions 13 to 28, It contains a helix-loop-helix dimerization domain (Prosite PS00038) and no adjacent basic domains. Schiffer-
Edmundson Helical Wheel Diagram (Schiffer et al., (1967) Biophys. J. 7: 121-135.
) Is used to predict a hypothetical amphipathic alpha helix between positions 53 and 68. Three hydrophobic amino acids, Val 55, Phe59 and Ile63, are aligned on the ipsilateral side of the helix, presenting a hydrophobic interacting surface, and three hydrophilic residues (Tyr53, Gln62).
And Ser64) are presented on the opposite side of the spiral. Proline residues that break the continuity of the alpha helix are not in this range. Thus, these structural features in the proteins of the present invention indicate that this protein may be a new member of the non-basic "helix loop helix" subfamily (HLH) of transcription factors.

The helix-loop-helix (HLH) family of transcription factors is involved in the control of different cell differentiation events such as neurogenesis, hematopoiesis, myogenesis and angiogenesis.
The HLH protein is found in all eukaryotes ranging from the yeast Saccharomyces cerevisiae to humans (Massari ME and Murre C. (2000) Molecular and
Cellular Biology, 20 (2): 429-440). HLH protein is DN
It binds A as a dimer, and different members of the HLH family bind as homodimers or heterodimers to other members of this family. Specific DN
The presence in cells of a large repertoire of different complexes capable of binding A sequence elements suggests that competition for DNA binding may play a regulatory role. Members of the helix-loop-helix (HLH) family of transcriptional control proteins contain 40-50 members containing a common structural element, two short amphipathic alpha helices separated by a variable length linker region (loop). Amino acid range of (Murre C et al., (1989) Cell 56: 777-783). This element is associated with the muscle-determining gene MyoD, c-myc (Davis RL et al., (1987) Cell 51).
: 987-1000), and a homologous region between the Drosophila achaete-scute complex (AS-C) (Villares R. and Cabrera CV (1987) Cell 50: 415-424) involved in neural determination. The HLH protein forms both homodimers and heterodimers by means of interactions between hydrophobic residues on the opposite faces of two helices, creating a four-helix bundle structure (Adrian R et al. (1993) Nature, 363: 38-
45; Ellenberger T et al. (1994) Genes Dev 8: 970-980). The alpha helix region generally has 15-16 hydrophobic residues at every 3rd and 4th position.
Each amino acid is long and each helix contains several conserved residues (Murre C et al. (1989)
Cell, 56: 777-783; Benezra R. et al., (1990) Cell, 61: 49-59). The HLH protein family is subdivided into two major groups, the so-called "bHLH" and "non-basic HLH" subfamilies. The bHLH family of proteins is
Containing a highly conserved highly basic region (known as bHLH structure) directly at the N-terminus in the first helix, mutagenesis experiments on the MyoD protein showed that this region was bHLH.
It has been confirmed to be responsible for sequence-specific binding to the E-box, a common DNA motif in proteins (Davis RL. Et al., (1990) Cell, 60: 733-746).
. Dimeric bHLH proteins (either homodimers or heterodimers in which both subunits contain a basic region) can bind to DNA. Generally, the bHLH protein is
Divided into two categories, class A consists of ubiquitously expressed proteins, which include mammalian E12 / E47 and fly da, while class B consists of proteins expressed in a more tissue-specific manner. Of mammalian MyoD and flies
Including AC-S. Most often, tissue-specific bHLH proteins preferentially heterodimerize with ubiquitous partners.

The non-basic HLH subfamily includes proteins lacking a basic region that are incapable of binding DNA but are capable of forming homo or heterodimers by virtue of their HLH motif. Indeed, the heterodimeric complex between abasic HLH and bHLH proteins fails to bind DNA and further negatively modulates bHLH protein-mediated transcriptional activation. This phenomenon was first demonstrated in the MyoD / Id control model (Bene
zra R. et al., (1990) Cell, 61: 49-59). The MyoD gene product, when introduced into a wide variety of differentiated cell types, can activate previously silent muscle-specific genes. The MyoD protein forms other bHLH proteins, such as E12 or E47, either homodimers or heterodimers, that bind the E-box common motif and activate myogenesis. Id gene conserved from amphibians to mammals (Wilson R et al. (1995) Mech. Dev. 49: 211-222; Sawai S et al. (1997) Mech.
Dev. 65: 175-185; Norton JD et al., (1998) trends in Cell Biology 8: 58-65.
) Lacks the basic region adjacent to the HLH motif, but MyoD, E12 or E14
It has been shown to be able to specifically dimerize with either of these and subsequently diminish the DNA binding capacity of the heterodimer. Moreover, overexpression of Id suppresses MyoD-dependent gene activation in in vivo transfection experiments. The Id protein either directly suppresses its activity by rendering the tissue-restricted bHLH protein non-functional, or
Or, if anything, block the ubiquitous bHLH protein, which is tissue-restricted.
It may function to block the formation of active heterodimers with bHLH (Norton J
D et al. (1998) trends in Cell biology 8: 58-65). The possibility that the Id protein acts as a dominant negative regulator that suppresses MyoD protein activity by forming a non-functional heterodimeric complex is significantly strengthened by the following findings in Drosophila. In Drosophila, development of the peripheral nervous system is associated with two structure-related bHLH proteins, AS-C and da.
It is positively regulated by ughterless (da), because the loss of either activity is due to the loss of sensory organ development. The extramacrochaetae Emc product belonging to the non-basic HLH subfamily has been shown to antagonize the activities of AS-C and da by forming a non-functional heterodimer with the bHLH protein ( Hillary M et al. (1990) Cell, 61: 27-38; Garrell J et al. (1990) Ce.
ll, 61: 39-48).

Human Id genes including human Id1, Id2, Id3 and Id4 have been identified and localized (reported by Norton JD et al. (1998) trends in Cell Biology 8: 58-65). The bHLH and Id proteins appear to be involved in the regulation of apoptosis. Differentiation and development of T and B lymphocytes in the immune system are positively regulated by the combination of the ubiquitous E protein and the lymphocyte-restricted bHLH protein. Disruption of gene expression from either class results in severe perturbation of T and B lymphocyte development (Bain G et al. (1997) Mol Cell Biol 17: 4782-4791;
Zhuang et al. (1996) Mol Cell Biol 16: 2898-2905). Cytostatic T thymocytes, Id
Overexpression of one gene causes a large amount of apoptosis (Kim D (1999) Mol Ce
ll Biol 19 (12): 8240-53). Overexpression of the Id1 gene product also causes apoptosis in cultured neonatal and adult cardiac myocytes (Tanaka K et al., (1998) JB.
iol Chem 273 (40) 25922-25928). Id1 and Id3 proteins are also required to support angiogenesis. Id protein expression is minimal on quiescent adult endothelial cells, but Id expression is upregulated on angiogenic endothelial cells. Partial loss of these proteins in Id1 ^+/- Id3 ^-/- double knockout mice impairs angiogenesis, resulting in resistance to tumor growth (Lyden D et al. (1999) Nature 401: 670-677). ). Furthermore, significant overexpression of Id1, Id2 and Id3 mRNA and protein levels has been found in patients with pancreatic cancer (Maruyama H et al. Am J Pathol (1999) 155 (3): 815-822.
). A further correlation between the Id1 gene upregulation and the aggressive phenotype of human breast cancer cells has been reported (Lin CQ et al. (2000) Cancer Res 60 (5): 1332-40). Therefore, the identification and cloning of members of the HLH family, particularly the non-basic HLH subfamily, has deepened our knowledge of the biological importance of the HLH transcription factor network, and in addition, the disease in disorders associated with HLH-mediated transcriptional dysregulation. It is necessary to provide knowledge and tools. SEQ ID NO: 394 and some of its proteins are likely to play a role in transcriptional activation, probably as members of the HLH family, preferably the abasic HLH subfamily. More specifically, the proteins of the invention appear to be able to antagonize the activity of bHLH family members by heterodimerization. A preferred polypeptide of the invention is a polypeptide comprising the amino acids of SEQ ID NO: 394 from positions 13-28, 53-68, and 13-68. Other preferred polypeptides of the present invention are fragments of SEQ ID NO: 394 having any of the biological activities described herein.

The ability of the proteins of the invention, or portions thereof, to dimerize, characteristic of the HLH family, can be assayed using any of the assays known to those of skill in the art. For example, especially bH
LH subfamily members, interacting protein partners include E12 and E47 (Murre C et al. (1989) Cell 56: 777-783), Max (Myc binding factor) (Elizab
eth M et al. (1991) Science 251: 1217), and Id (Benezra C et al. (1990) Ce.
ll 61: 49-59) as described for the identification of several HLH transcription factors, such as cDNA
It can be identified using screening of expression libraries. Alternatively, the helix-loop-helix motif in the protein of the present invention is referred to as a "bait protein" in a well of the well-known yeast two-hybrid method from a cDNA library derived from a different tissue or cell type of a given organism and interacting in vivo. It can be used by those skilled in the art to identify protein partners. Alternatively, the proteins of the invention or portions thereof may be used by those skilled in the art of mammalian cell transfection experiments. When fused to an appropriate peptide tag, such as the <His> ₆ tag in a protein expression vector and introduced into cultured cells, this expressed fusion protein can be expressed as a potential intergenic peptide by using an anti-tag peptide antibody. Can be immunoprecipitated with a working protein. This method can be selected for identification of the association partners or confirmation of the results obtained by other methods such as those described above. The purpose of the present invention is to overexpress the protein of the present invention by any means known to those skilled in the art, thereby abrogating the transcription of the gene, preferably the HLH regulator mediated transcription in vitro or in vivo. Thus, it relates to compositions and methods using the proteins of the invention or portions thereof. The proteins of the invention, or portions thereof, are capable of inducing apoptosis of certain cell types under physiological or pathological conditions. In a preferred embodiment, an apoptosis-inducing effective amount of an apoptotic polypeptide is added to the in vitro culture of mammalian cells. In another preferred embodiment, the apoptotic polypeptide is expressed under the control of a promoter that can be activated under the correct conditions. In particular, such conditional expression, which is an on-demand form of apoptotic polypeptide, eliminates unnecessary cells, for example, in applications where such cells are used for cell therapy purposes and become unnecessary. It is extremely useful for Another example of application is in the case of expression under the control of a promoter which is active after infection with a given microorganism, thus resulting in the death of only infected cells. Further, the proteins of the invention, or portions thereof, may be used to diagnose, treat and / or treat disorders in which apoptosis would be beneficial, including, but not limited to, disorders associated with abnormal cell proliferation such as those described below. Or it may be useful in prevention.

In yet another embodiment, the protein of the invention, or a portion thereof, may be administered to cancer and hyperaldosterone, hypoadrenocorticism using any of the methods and / or techniques described herein. (Addison's disease), hyperthyroidism (Graves' disease), hypothyroidism, colorectal polyps, gastritis, gastric and duodenal ulcers, ulcerative colitis, and neurodegeneration such as Crohn's disease, Parkinson's disease or Alzheimer's disease. It may be used for the diagnosis, treatment and / or prevention of disorders associated with overexpression of HLH protein, such as disorders associated with abnormal cell differentiation, proliferation or degeneration, including disorders. Furthermore, the protein of the present invention or a part thereof is
It can be used to assess disease progression and efficacy of clinical treatment. The proteins of the invention or portions thereof can also be used as molecular targets in anti-angiogenic drug design. Inhibition of protein expression can be achieved by numerous means known to those of skill in the art, including those described in this application. For example, antisense nucleotides or triple helix strategies can be developed to block protein synthesis. Alternatively, the expressed proteins of the present invention are described in Peverali FA et al., (1994) EMBO J
13: 4291-4301; Barone MV et al., (1994) Proc. Natl. Acad. Sci. USA 91:49.
85-4988; and Haza ET et al., (1994) J. Biol. Chem. 269: 2139-2145 using techniques known to those of skill in the art using specific monoclonal antibodies. You can harmonize. [Protein of SEQ ID NO: 466 (Internal Designation Number 184-4-2-0-D3-CS)] The protein of SEQ ID NO: 466 is overexpressed in the liver and has RING type (C3HC4) (Pfam signature from positions 41 to 81, It is encoded by the cDNA of SEQ ID NO: 225 exhibiting a Zn finger motif that is the Prosite signature from positions 56-65) and a B-box Zn finger motif (pfam signature from positions 110-153). Furthermore, the proteins of the invention are predicted to have nuclear localization. It is believed that the protein of SEQ ID NO: 466 or a portion thereof is a zinc binding protein, preferably capable of binding nucleic acids, more preferably transcription factors. A preferred polynucleotide of the invention is a polypeptide comprising the amino acids of sequence 466 from positions 41-81 (RING Zn finger protein) and positions 110-153 (B-box domain). Other preferred polypeptides of the present invention are fragments of SEQ ID NO: 466 having any of the biological activities described herein.

[Protein of SEQ ID NO: 267 (Internal Designation No. 116-111-1-0-H9-CS)] The protein of SEQ ID NO: 267 encoded by the extended cDNA of SEQ ID NO: 26 is 185
The Emotif Zn finger domain from ~ 202 position, C2H2 type, is assumed to be localized in the nucleus. It is believed that the protein of SEQ ID NO: 267 or a portion thereof is a zinc binding protein, preferably capable of binding nucleic acids, more preferably transcription factors. A preferred polypeptide of the invention is a polypeptide comprising the amino acids of sequence 267 from position 185-2202. Other preferred polypeptides of the present invention are fragments of sequence 267 that have any of the biological activities described herein. [Protein of SEQ ID NO: 277 (Internal Designation No. 160-103-1-0-F11-CS)] The protein of SEQ ID NO: 277 encoded by the extended cDNA of SEQ ID NO: 36 is 140
It presents the pfam DHHC Zn finger domain from position ~ 204. It is believed that the protein of SEQ ID NO: 277 or a portion thereof is a zinc binding protein, preferably capable of binding a nucleic acid, more preferably a transcription factor. A preferred polypeptide of the invention is a polypeptide comprising residues of sequence 277 from positions 140-204. Other preferred polypeptides of the present invention are fragments of sequence 277 that have any of the biological activities described herein. [Protein of SEQ ID NO: 272 (Internal designation number 145-25-3-0-B4-CS)] The protein of SEQ ID NO: 272 encoded by the extended cDNA of SEQ ID NO: 31 shows homology with many zinc-binding proteins. Show. Furthermore, the protein of the present invention is 87
The pfam RING Zn finger signature from position 129 is presented. The protein of SEQ ID NO: 272 has a variant, ie the protein of SEQ ID NO: 273 encoded by the extended cDNA of SEQ ID NO: 32 and is believed to have the same function and utility. It is believed that the protein of SEQ ID NO: 272, or a portion thereof, is a zinc binding protein, preferably capable of binding nucleic acids, more preferably transcription factors. A preferred polypeptide of the invention is a polypeptide comprising the amino acids of sequence 272 from positions 87-129. Other preferred polypeptides of the present invention are fragments of sequence 272, which have any of the biological activities described herein. <Hydrolytic Enzymes and Inhibitors> The present invention provides a protein of the present invention having hydrolytic activity, referred to herein as HYP, such as those described in this section, and a hydrolytic domain as shown in Table VI. Compositions and methods that include, or a portion thereof, preferably a fragment containing a hydrolysis domain, or derivative thereof.

The present invention relates to methods and compositions using HYP or fragments thereof for hydrolyzing one or more substrates, alone or in combination with other substrates.
For example, a protein of the invention, or a portion thereof, is added to a sample containing substrate (s) under conditions that allow it to hydrolyze and catalyze the hydrolysis of the substrate (s). .. The hydrolyzed substrate is then detected using standard methods known to those of skill in the art. The proteins of the invention, or portions thereof, may also be used as "cocktails" with other hydrolases such as other peptidases, eg, US Pat.
It may be added to the sample to decontaminate the surgical instrument using the method described in 5,489,531. The advantage of using a cocktail of hydrolases is that they can hydrolyze a wide range of substrates without necessarily knowing the specificity of each enzyme. The use of hydrolase cocktails also protects the sample from a wide range of unknown contaminants from vast numbers of sources. Alternatively, HYP, or a portion thereof, alone, using techniques known to those of skill in the art to create an affinity chromatography column for the purpose of substrate removal,
Alternatively, it may be bound to a chromatographic support in combination with other hydrolases. Immobilization facilitates removal of the enzyme from the batch product, as well as subsequent reuse of the enzyme. Immobilization of the enzyme or part thereof can be achieved by adding a cellulose binding domain to the protein, for example by modification of the DNA sequence encoding the protein or part thereof. Other methods of immobilization are also available to those of skill in the art,
It will be understood that this is explained in the published literature. Alternatively, similar methods can be used to identify new substrates. In other embodiments, HYP or a portion thereof can be used to identify or quantify a given substrate in a biological sample. In a preferred embodiment, HYP or a portion thereof is catalytically inactivated using any of the methods known to those of skill in the art, including those that produce mutant enzymes, recombinant enzymes, or chemically inactivated enzymes. (Ie it can bind to a given substrate but cannot be hydrolyzed). The catalytically inactive protein of the invention is then incubated with an aliquot of the biological sample under suitable conditions for binding the inactive enzyme to the substrate. The presence of eubacteria in the biological sample is then detected, or the bound enzyme is detected to assess bacterial load. In another preferred embodiment, HYP or a portion thereof is used in an assay and diagnostic kit for the identification and quantification of a substrate in a biological sample. These assays are based, for example, on enzyme-linked immunosorbent assays (ELISA) or other techniques known to those of skill in the art.

In addition, HYPs or portions thereof can be used to identify enzyme inhibitors for mechanistic and clinical applications using screening based on standard assay methods such as those described above. . Such inhibitors can then be used to identify or quantify HYP present in a sample, and to diagnose, treat, or prevent disorders in which protein activity is undesirable and / or harmful. [Protein of SEQ ID NO: 400 (Internal designation number: 160-54-1-0-F7-CS)] The protein of SEQ ID NO: 400 encoded by the cDNA of SEQ ID NO: 159 was prepared by software TopPred II (Claros and von Heijne, CABIOS applic Notes, 10: 685-68
6 (1994)) and presents two putative transmembrane domains surrounding the amino acids at positions 50-70 and 127-147. It also exhibits the Prosite carboxypeptidase zinc binding domain signature PS00133 at positions 117-127.
It uses psort software (Nakai K and Horton P, Trends Biochem Sci. 1999 J
an; 24 (1): 34-6), it is predicted to be localized in the nucleus with high probability (73.9%). After all, it is specifically expressed in fetal brain and shows no homology with known proteins. Carboxypeptidase enzymes hydrolyze the terminal amino acids of proteins or peptides. A new family of carboxypeptidases, localized in the nucleus and having carboxypeptidase-dependent transcriptional activity, has recently emerged. Its first member, AEBP1, was previously identified as the 3T3 preadipocyte factor involved in suppression of aP2 gene expression. AEBP1 means "AE-1 binding protein", AE-1 is a regulatory element of adipose P2 gene (aP2), a gene involved in triglyceride metabolism and involved in adipocyte activation. . Its expression disappears during adipocyte differentiation (He GP et al., Nature 378: 92-96 (19
95). AEBP1 is a differentiation of osteoblastic cell lines (Ohno I et al., Biochem Biophys Res Commun.
1996 Nov 12; 228 (2): 411-4) and vascular smooth muscle cells (Layne MD et al., J. Biol. C).
hem: 273: 15654-15660 (1998)) was later shown to play a similar role. It was proposed that AEBP1 act as a negative transcription factor by cleaving proteins involved in transcription, which is a novel feature of transcriptional regulation. Recent experimental results further indicate that its transcriptional activity is attenuated by binding to G protein subunits (Park JG et al., EMBO J. 1999 Jul 15; 18 (14): 4004-12) and stimulated by DNA binding. Things (Muise AM and RoHS, Biochem J. 1999 Oct 15; 343
Pt 2: 341-5).

The protein of SEQ ID NO: 400 is believed to be involved in cell signaling, nuclear transcriptional activity and several different cell types (including but not limited to neurons), especially those found in the developing brain. ing. Preferred polypeptides of the invention are polypeptides having any of the biological activities described herein. One embodiment of the invention relates to compositions and methods using the proteins of the invention or portions thereof as markers for specific cell compartments (especially the nucleus) and / or tissue types (especially fetal brain). For example, the proteins of the invention or portions thereof can be used to produce specific antibodies that allow visualization of nuclear structure by methods known to those of skill in the art. Similarly, an antibody raised against a protein of the invention may be present at a particular developmental stage (eg, fetal) and / or predetermined because the protein of the invention is specifically expressed in fetal brain tissue. Can be used to identify the tissue type (eg, brain) of. Antibodies and antisera can also be used to inhibit undesired carboxypeptidase activity in in vitro experiments and cell culture, as well as in biological samples and in vivo. Alternatively, the quantitative analysis or detection of the protein of the invention, or the nucleic acid encoding the protein, can be carried out by any of the other techniques known to those skilled in the art. In another embodiment, the proteins of the invention can be used to target a heterologous compound (polypeptide or polynucleotide) to the developing brain and / or cell nucleus. For example, a chimeric protein consisting of a protein of the present invention recombinantly or chemically fused with a therapeutic protein or polynucleotide of interest has a therapeutic protein / polynucleotide in the above cells / target tissues (nucleus, fetal brain). Allows for specific delivery. In another embodiment, the invention relates to methods and compositions that employ the proteins of the invention or fragments thereof to hydrolyze one or several substrates, alone or in combination with other substrates. The ability of the protein to hydrolyze a particular substrate may be determined by standard assays such as those described by Slusher et al. It can be easily determined by performing a hydrolysis reaction using other known techniques. Possible substrates are substrates that contain peptide bonds, particularly C-terminal peptide bonds. Such substrates include, but are not limited to, polypeptides, folic acid and the like (eg, methotrexate). For example, a protein of the invention, or a portion thereof, is added to a sample containing substrate (s) under conditions that allow it to hydrolyze and catalyze the hydrolysis of the substrate (s). .. The hydrolyzed substrate is then detected using standard methods known to those of skill in the art.

In a preferred embodiment, the proteins of the invention, or portions thereof, can be used to modulate transcriptional activity of cells and thereby cell differentiation. In particular, nuclear carboxypeptidases are involved in repression of transcription associated with differentiation,
Therefore, increased activity or expression of the protein can be used to suppress differentiation. The ability to suppress differentiation is, for example, to keep cultured cells in an undifferentiated state until it is necessary to give rise to a given cell type during the culture of undifferentiated pluripotent cells (eg transplantation) , With several uses. The level of protein activity or expression includes introducing into the cell a polynucleotide encoding the protein, administering the protein itself to the cell, or administering to the cell a compound that increases protein activity or expression. It can be increased by any of several methods.
Alternatively, the proteins of the invention can be repressed, thereby enhancing cell differentiation. The ability to promote differentiation has numerous uses, including the treatment or prevention of cancer, as cancer cells are often in a relatively undifferentiated state, and cell differentiation is generally associated with growth arrest. In another embodiment, the proteins of the invention or portions thereof are for diagnosing, treating and / or preventing disorders in which the presence of substrates such as excess proteins or peptides is undesirable or detrimental. Can be used for Such disorders include neurodegenerative disorders such as cancer, Parkinson's disease and Alzheimer's disease,
And diabetes, but is not limited thereto. In other embodiments, the proteins of the invention, or portions thereof, can be used for the identification or quantification of a given substrate (eg, peptide, folic acid, or methotrexate) in a biological sample. In another preferred embodiment, the proteins of the invention or portions thereof are used in assay and diagnostic kits for the identification and quantification of substrates in biological samples. In the most preferred embodiment, the protein of the invention or a portion thereof can be used in rescue therapy after highly toxic dose methotrexate treatment in cancer chemotherapy. Many carboxypeptidases are capable of cleaving the C-terminal glutamate moiety from folic acid and its analogs such as methotrexate. The major role of reduced folate as a coenzyme in a number of biological pathways, including those that direct DNA synthesis via pyrimidines and pilins, is to make folic acid a target molecule for chemotherapy. Tumor cells grow rapidly and therefore have a high rate of nucleic acid synthesis. Folic acid depletion status is
It has a cytotoxic effect mainly in replicating tissues and can suppress the growth of tumors that require high amounts of folic acid. Many carboxypeptidases can directly remove folic acid by removing its glutamate moiety by hydrolysis. In cancer chemotherapy, methotrexate (4-amino -N ¹⁰ - methyl - pteroyl - glutamate) is generally the reduction of folic acid to the catalyst, is essential for the biosynthesis of all the folate coenzymes, biological Used to eliminate the pool of reduced folate by inhibiting dihydrofolate reductase (DHFR), which results in the active tetrahydrofolate form. Accordingly, the proteins of the invention, or portions thereof, are broadmann, the disclosures of which are incorporated herein by reference, Widemann et al.
.Assoc. Cancer Res.- 1995, 36, p232) and Chabner et al., (Chabner B.
Et al., Nature-1972, 239, p395-397) and can be used in rescue therapy after high dose toxicant measures. The basic of this strategy, methotrexate hydrolysis, low about 100 times more active as an inhibitor of DHFR 4-Amino -N ¹⁰ - methyl - a pteroyl salt is to produce.

In another preferred embodiment, the protein of the invention, or a portion thereof, comprises, but is not limited to, autoimmune diseases and chronic inflammatory diseases such as rheumatoid arthritis,
And can be used in enzyme / prodrug strategies to treat several pathological conditions, including those that are treated with drugs with particularly severe side effects, including cancer chemotherapy. These side effects can be explained mainly by the fact that the in vivo selectivity of the drugs used is too low (for example, the poor selectivity between tumor and normal cells of most anticancer drugs is well known and Tissue toxicity is dose limiting). In the first phase of one example of such a protocol, a conjugate of a protein of the invention or a portion thereof and an antibody of a tissue-specific antigen (eg, a tumor-specific antigen in cancer chemotherapy) is administered. A relatively non-toxic compound is administered to the patient after some time to clear the residual enzyme conjugate from the blood. This non-toxic compound is
It is a substrate of the protein of the present invention, and is converted into a substantially more toxic compound by the present protein. Thus, for the pre-targeted administration of the proteins of the invention, when a non-toxic compound is administered, the toxic compound is produced only in the vicinity of the cells targeted by the fusion protein. This biphasic method is referred to as antibody-directed enzyme-prodrug therapy (ADEPT) and is described in Melton et al. (Melton R. et al.
-J. Natl. Cancer Inst.- 1996, 88, p153-165).
Alternatively, the first phase may be replaced by gene therapy resulting from the neosynthesis of a protein of the invention or part thereof by cells from the target tissue, which is called gene-dependent enzyme / prodrug therapy (GDEPT). . These two methods (ADEPT and
Another advantage of GDEPT) is that a single enzyme molecule can activate multiple prodrug molecules. [Protein of SEQ ID NO: 242 (internal designation number 119-003-4-0-C2-CS)] The protein of SEQ ID NO: 242 encoded by the cDNA of SEQ ID NO: 1 is a protein of the M20 metallopeptidase family (EC 3.4.17.X. ) Is homologous to the protein. The proteins of the invention are overexpressed in the spinal cord and brain. The M20 metallopeptidase family of proteins are all peptidases (ie, enzymes capable of hydrolyzing peptide bonds) and all are exopeptidases, which means that they can hydrolyze the terminal amino acids of a protein or peptide. Members of the M20 peptidase family are glutamate carboxypeptidases that are capable of releasing C-terminal glutamic acid residues from a wide range of N-acyl groups, including peptidyl, aminoacyl, benzoyl, benzyloxycarbonyl, folyl, and pteroyl groups, by hydrolysis. Are involved in protein catabolism. The M20 carboxypeptidase is either a monomer or a homodimer (ie, two identical proteins constructed to form the enzyme). To be active, metallopeptidases must associate with metal cofactors (zinc or cobalt depending on the enzyme). The most closely studied M20 family of carboxypeptidases is Pseudomonas sp. (RS-16 strain)
It is carboxypeptidase G2 (CPG2) (EC 3.4.17.11), which is a bacterial enzyme of origin.
CPG2 is a dimeric zinc carboxypeptidase that cleaves the C-terminal glutamate moiety from several molecules.

The protein of SEQ ID NO: 242 contains the pfam signature of the M20 peptidase (positions 107-451). The protein of SEQ ID NO: 242 also specifically interacts with the metal cofactor M20.
It contains some amino acids that are conserved throughout the protease family. Preferred polypeptides of the invention are present in 80% or more of the M20 peptidase family members and are highly conserved amino acids 133, 135, 149, 163, 200, 201.
Amino acids 139, 157, 162, 16, 367 and / or containing 262 and / or 262 and / or present in 60% or more of the members of the M20 peptidase family
Including 377. Of particular interest are amino acids 133, 166, 201 and 262 which, because of their homology, are probably involved in the interaction with metal cofactors. Thus, the protein of SEQ ID NO: 242 or a portion thereof is a M20 family peptidase, preferably a carboxypeptidase, more preferably a metal carboxypeptidase. Other preferred polypeptides of the present invention are fragments of SEQ ID NO: 242 having any of the biological activities described herein. Determining carboxypeptidase activity on a particular substrate is described by Slusher et al.
easily obtained by carrying out the hydrolysis reaction using standard assay methods such as those described by sher et al.-Prostate-2000, 44 (1): 55-60), or other techniques known to those of skill in the art. be able to. Possible substrates are substrates that contain peptide bonds, particularly C-terminal peptide bonds, and more specifically C-terminal glutamic acid. Such substrates include, but are not limited to, polypeptides, folic acid and the like (eg, methotrexate). In embodiments, the proteins of the invention, or portions thereof, may be used to develop assay tools to identify tissues of the invention as they are overexpressed in brain and spinal cord tissues. In other embodiments, the proteins of the invention or portions thereof are used to diagnose, treat and / or prevent disorders in which the presence of substrates such as excess proteins is undesirable or detrimental. it can. Such obstacles are cancer,
Includes, but is not limited to, neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease, and diabetes. In a most preferred embodiment, the protein of the invention or a portion thereof can be used in rescue therapy following a highly toxic dose of methotrexate in cancer chemotherapy. The M20 peptidase family of enzymes is capable of cleaving the C-terminal glutamate moiety from folic acid and its analogs such as methotrexate. The major role of reduced folate as a coenzyme in a number of biological pathways, including those that direct DNA synthesis via pyrimidines and pilins, is to make folic acid a target molecule for chemotherapy. Tumor cells grow rapidly and therefore have a high rate of nucleic acid synthesis. A folate depleted state has a cytotoxic effect primarily in replicating tissues and can even inhibit the growth of tumors that require high amounts of folate. Enzymes of the M20 peptide family can remove their glutamate moiety directly from folate by hydrolysis. In cancer chemotherapy,
Methotrexate (4-amino -N ¹⁰ - methyl - pteroyl - glutamate) is generally the reduction of folic acid to the catalyst, it is essential for the biosynthesis of all the folate coenzymes, tetrahydrofolic acid with a biologically active It is used to eliminate the pool of reduced folate by inhibiting the formation of dihydrofolate reductase (DHFR). Therefore,
The protein of the present invention or a part thereof, Widemann et al., (Widemann B. et al., -Proc. Am. Assoc. Cance, the entire disclosures of which are incorporated herein by reference.
r Res.-1995, 36, p232) and Chabner et al., (Chabner B. et al.,-Nature-
1972, 239, p395-397) and can be used in rescue therapy after high dose toxicant measures. The basis of this strategy is that the hydrolysis of methotrexate produces 4-amino-N ¹⁰ -methyl-pteroyl-glutamic acid which is about 100 times less active as an inhibitor of DHFR.

In another preferred embodiment, the protein of the invention, or a portion thereof, comprises, but is not limited to, autoimmune diseases and chronic inflammatory diseases such as rheumatoid arthritis,
And can be used in enzyme / prodrug strategies to treat a number of pathological conditions, including those associated with particularly severe side effects, including cancer chemotherapy. These side effects can be explained mainly by the fact that the in-vivo selectivity of the drugs used is low (for example, the poor selectivity of most anti-cancer agents between tumor and normal cells is well known and Toxicity is dose limiting). In the first phase of one example of such a protocol, a conjugate of a protein of the invention or a portion thereof and an antibody of a tissue-specific antigen (eg, a tumor-specific antigen in cancer chemotherapy) is administered. A relatively non-toxic compound is administered to the patient after some time to clear the residual enzyme conjugate from the blood. This non-toxic compound is a substrate for the protein of the invention and is converted by the protein into a substantially more toxic compound. Thus, when a non-toxic compound is administered for pre-targeted administration of the protein of the invention, the toxic compound is only produced in the vicinity of the cells targeted by the fusion protein. This biphasic method is called antibody-directed enzyme prodrug therapy (ADEPT), and the method is described in Melton et al., (Melton R. et al., J. Natl. Cancer Inst.-1996, 88, p153- 165). Alternatively, the first phase may be replaced by gene therapy resulting from the neosynthesis of a protein of the invention or part thereof by cells from the target tissue, which is called gene-dependent enzyme / prodrug therapy (GDEPT). . These two methods (ADEPT
And GDEPT) is that a single enzyme molecule can activate multiple prodrug molecules. [Protein of SEQ ID NO: 401 (internal designation number 160-88-3-0-A8-CS.corr)] The protein of SEQ ID NO: 401 encoded by the cDNA of SEQ ID NO: 160 is assumed to be human palmitoyl protein thioesterase-2 ( PPT2) (EC 3.1.2.22) (Genbank accession number AF020543), a eukaryotic (C. elegan)
s and rodents) and shows homology with palmitoyl protein thioesterase-1 (PPT1) (Genbank accession number L42809). SEQ ID NO: 16
The product with 0 cDNA is shorter than human PPT2 (280 amino acids vs. 308 amino acids),
It has a gap between position 174-203 of protein PPT2. The protein of SEQ ID NO: 401 has a variant that is the protein of SEQ ID NO: 402 encoded by the cDNA of SEQ ID NO: 161 and is considered to have the same function and utility.

[0151]   PPT1 (E.C. 3.1.2.22) is a well-known protein and is found in mice and rats.
Widely conserved among bovine and human species (Swissprot accession number P50897).
It is useful for removing fatty acids from modified cysteine residues in degraded proteins.
A lysosomal enzyme that functions in different ways (Hofmann S.L. et al., Neuropediatrics, 28:
 27-30 (1997). For example, PPT1 is a heterodimeric versus G-protein H-ra in vitro.
Catalyzes deacylation of s and alpha subunits (Camp L.A., J. Biol.
 Chem., 268: 22566-22574 (1993) and 269: 23212-23219 (1994)). PPT1
Deacylation by can be essential for complete digestion of the modified polypeptide. Fruit
In doing so, palmitoylation may increase protection against proteolytic digestion.
Has been proven. Salivary mucosa glycoprotein (Slomiany B. L., Biochem. Biophys.
 Res. Commun., 151: 1046-1053 (1988)), and chemically acylated ha.
Zytoxin phospholipase A2 (Diaz, R.E., Biochem. Biophys. Acta, 830: 52-58 (
1985)) both are more resistant to treatment with proteinases than their deacylated forms
There is. Mutations in the PPT1 enzyme are associated with inherited neurodegenerative disorders, neuronal ceroid
It has been shown to be responsible for the pediatric form of pofustinosis (INCL) (Vesa et al. Nat
ure, 376: 584-587 (1995)).   Recently, Soyombo and Hofmann (J. Biol. Chem, 272: 27456-27463, (1997))
Report a second lysosomal thioesterase, PPT2, that shares 20% identity with PPT1
I told you. The PPT2 enzyme is also probably involved in lysosomal thioester catabolism
However, it has a different substrate specificity from PPT1. About the substrate specificity of PPT2
Although unknown, this enzyme is a palmitoylated model such as palmitoyl CoA.
High activity against substrate. PPT2 is similar to H-Ras and albumin-like PPT1.
Hydrolyzes acyl-cysteine bonds in protein substrates routinely used in assays
do not do. This finding suggests that both enzymes have endogenous palmitoyl thioesterase activity.
, But shows that the “leaving group” recognized by the enzyme may be different.
Incite. One possibility is that PPT2 recognizes palmitoylated protein substrates,
These substrates are different from those recognized by PPT1. Second acceptable
Potentially, PPT2 is a new lipid thioester substrate not derived from acylated proteins
Is to recognize. Aguado et al. (Biochem J., 341: 679-689, (1999))
, PPT2 was demonstrated to be an acyl thioesterase. But they
No distinction can be made between the tellase (thioesterase) and lipase activities. PPT2 is
Sill chain C_{14: 0}> C_{16: 0}   Extremely high S-thioesterase activity, acyl chain C _{14: 1} > C_{20: 4}≈ C_{16: 1}≈ C_{18: 0}≈ C_{12: 0}> C_{18: 2}≈ C_{18: 3}> C_{22: 1}≈ C_{18: 1}≈ C_{20: 0}About
Moderate activity, acyl chain C_{10: 0} And C_{22: 0}Shows low activity with
Rani C_{24: 0}, C_{8: 0}, C_{6: 0}, C_{4: 0} And C_{2: 0}Shows no activity at all.
PPT2 has a broader spectrum of action than PPT1, but both have shorter acyl chains (12 carbons or less).
Prefer longer acyl chains (12 or more carbons) than below). Aguado et al.
) Also showed detailed characterization of the PPT2 gene product. Putative 302 residues PPT2 and
And the protein of the present invention have a hydrophobic leader peptide (protein of the present invention
Signal peptide with its predicted cleavage site at position 34 of the quality)
Is a secretory glycoprotein. Both proteins have N-terminal
It presents two motifs from the located 108-121 positions. One motif is
Liglyceride lipase (from position 110-121), the other is eukaryotic thio (Cys)
Common to Rotease (from position 108-121). Triglyceride lipase
It is a lipolytic enzyme that hydrolyzes the ester bond of lyglyceride. All of these
The most conserved region of all proteins is the Gly-Xaa-Ser-Xaa-Gly conserved motif.
Concentrated on serine residues located in the erfs. PPT2 protein and the invention
The protein replaces the first glycine residue in the motif with a cysteine residue (115
Position) but with one mismatch in either of the consensus
Has been described (Blow D., Nature, 343: 694-695 (1990)). PPT2
And the protein of the present invention contains a eukaryotic thio (Cys) in the same region as the lipase motif.
It contains a motif common to the active sites of proteases, but is located at position 5 in the pattern.
It has a leucine residue (position 113) instead of lysine. In addition, the estimated PPT2
The no acid sequence is a C-terminal growth factor and cytokine receptor from position 171-186.
It shows a motif common to the family, but this does not exist in the proteins of the present invention.
Yes.

Aguado et al. (Supra) found that PPT2 is expressed in immune system cells as an approximately 42 kDa protein present in cell extracts and supernatants and transcribed as at least five different transcripts. . The PPT2 gene is located in the class III region of human MHC, which contains several genes that encode proteins with potential roles in the immune system and inflammation. In addition, Aguado et al. (Supra) showed that very large amounts of PPT2 are secreted. However, this is consistent with intracellular activity because secreted proteins can be internalized into cells via receptors and act on targets located in intracellular organelles. This mechanism has been described for secreted PPT1 that is internalized into cells by the mannose-6-phosphate receptor and can act within lysosomes (Verkruyse and Hofmann, J. Biol. Chem., 271: 15831- 15836, (1996)),
Soyombo and Hofmann (J. Biol. Chem, 272: 27456-27463, (1997))
We reported that PT2 binds to the mannose-6-phosphate receptor. Palmitoylation is a post-translational modification of proteins in which the most common cellular fatty acids (ie, palmitic, stearic, and oleic acids) are attached to the side chains of cysteine residues via high-energy thioester bonds. Mean (Bizzoz
ero, OA et al., Neurochem. Res., 19: 923-933 (1994); Casey PJ, Scienc.
e, 268: 221-225 (1995)). It is now known that many proteins of diverse origin, structure and function can be modified by such fatty acids and attached to the inner surface of the plasma membrane where they function optimally (Casey PJ, Science, 26
8: 221-225 (1995)). Membrane anchoring is a necessary process for the diverse cellular functions of these modified proteins, including signal transduction, vesicle trafficking and maintenance of cellular architecture. Almost all tissues and subcellular organelles contain a characteristic set of palmitoylated proteins. The protein of the present invention is overexpressed in the brain. Recently, it has been discovered that a significant number of functionally related nervous system proteins, including ion channels, neurotransmitter receptors, signaling components and cell adhesion molecules, are palmitoylated. Although the nervous system is no exception to this rule, both the number of modified proteins in this tissue and the dynamic nature of protein palmitoylation are crucial factors in the regulation of biological processes where this modification is important, Furthermore, it is suggested that the addition or removal of fatty acids plays a role in controlling the activity of these proteins rather than defining their function.

The protein of SEQ ID NO: 401 or a part thereof is a hydrolase, preferably acts on an ester bond, more preferably a thioester hydrolase,
More preferably, it is an acyl thioesterase and is involved in fatty acid metabolism, maintenance of cell vesicle transport and cell assembly, cell proteolysis, endocytosis, signal transduction, lysosomal storage, cell proliferation and differentiation, immune and inflammatory response. To do. The enzyme substrate is a compound containing an ester bond, preferably a thioester bond, and preferably an acylthioester bond. A preferred polypeptide of the invention is a polypeptide comprising the amino acids of sequence 401 from positions 108-121 and 110-121. Other preferred polypeptides of the present invention are fragments of sequence 401, which have any of the biological activities described herein. The hydrolytic activity of the protein of the present invention, or a portion thereof, is determined by Smith et al., Biochem. J., 212: 155 (
1983), Spencer et al., J. Biol. Chem., 253: 5922 (1978) and Aguado et al., (Supra) or using any of the assays known to those of skill in the art, including those described in US Pat. Can be tested. In other preferred embodiments, the protein of the invention or a portion thereof is, for example,
It can be used to diagnose, treat and / or prevent disorders in which the presence of a substrate is undesirable or harmful. Such disorders include, but are not limited to, the pediatric form of neuronal ceroid lipofuscinosis (INCL) and lysosomal disorders. For diagnostic purposes, expression of the proteins of the invention can be studied using Northern blot, RT-PCR, immunoblotting methods described herein and compared to expression in control individuals. For prophylactic and / or therapeutic purposes, expression of the proteins of the invention can be enhanced using any of the gene therapies described herein or known to those of skill in the art. In addition, the proteins of the invention, or portions thereof, can be used to identify inhibitors for mechanistic and clinical applications. Such inhibitors then identify or quantify the proteins of the invention in the sample, as well as lysosomal disorders, neuronal ceroid lipofuscinosis (INCL) pediatric types, neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. It can be used for the diagnosis, treatment or prophylaxis of disorders in which the hydrolytic activity of said proteins is undesirable and / or harmful, including but not limited to inflammatory and immunodeficiency diseases including allergies and leukemias.

Another object of the present invention is to recombinantly or chemically fuse a fragment of the protein of the present invention to a heterologous polypeptide or polynucleotide so that the heterologous compound, which is either a polypeptide or a polynucleotide, is lysosomal. And compositions targeting to. A preferred fragment is a fragment of the protein of the invention or a part thereof, Vitale et al., Mol. Cell. Biol, 20: 7342-52 (20
00), Blagoveshchenskaya et al., J. Biol. Chem., 273: 2729-37 (1998) and Ko.
Target signals for lysosomes may be included, such as those described by rnfeld, FASEB J., 1: 462-8 (1987). Such heterologous compounds can be used to modulate lysosomal activity. For example, they can be used to induce and / or prevent lysosomal proteolysis. Furthermore, antibodies that bind to the protein of the invention or a portion thereof can be used to detect lysosomes using any of the techniques known to those of skill in the art. In yet another embodiment, the invention relates to methods and compositions using a protein of the invention or a portion thereof as a marker protein for selectively identifying tissue, preferably brain tissue, eg, a protein of the invention or Some of them are
Techniques known to those of skill in the art, including those described herein, can be used to synthesize specific antibodies. Such tissue-specific antibodies can then be used to identify tissues of unknown origin, such as differentiated tumor tissues that have metastasized to forensic samples, different body sites, etc., or use immunochemistry to identify different tissues in a tissue cross section. Can be used to classify types. Other embodiments of the invention are described in U.S. Patents 5,955,650, 5,945,585 and 5,807,893.
Methods and compositions for using the proteins of the invention or portions thereof to modify plant lipid compositions using any of the assays known to those of skill in the art, including those described in. In fact, plant lipids have a variety of nutritional uses, and many recent research efforts have explored the role that saturated and unsaturated fatty acids play in reducing the risk of coronary heart disease. In the past, monounsaturates, in contrast to saturates and polysaturates, were not considered to affect serum cholesterol and the risk of coronary heart disease. A recent clinical study in humans found that a diet high in monounsaturated fat and low in saturated fat reduced "bad" (low density lipoprotein) cholesterol and "good" (high density lipoprotein) cholesterol. Suggesting that they can be maintained ((Mattson et al., Journal of Lipid Research, 26
: 194-202 (1985)).

In yet another embodiment, a protein of the invention, or a portion thereof, may be supplemented with enzyme replacement therapy (Neufeld EF, Annu. Rev. Biochem. 60 257-280 (1991),
Brady RO et al., J. Inher. Metab. Dis. 17: 510-519 (1994)), or bone marrow-derived microglial cells, cross the blood-brain barrier and theoretically correct neuronal metabolic defects. (Krivit W.,
Cell transplant., 4: 385-392 (1995), Bone marrow transplant (Hoogerbrugge PM et al., L
ancet, 345: 1398-1402 (1995)). The proteins of the invention, or portions thereof, may also be genetically engineered into transplanted cells using techniques known to those of skill in the art (Salvetti A. et al. Br.Med.J. 51: 106-122 (1995)) or It can be used for neural progenitor cell transplantation (Snyder EY, Nature, 374: 367-370 (1995)). [Protein of SEQ ID NO: 254 (Internal Designation No. 106-006-1-0-E3-CS)] Angiogenin is a member of the pancreatic RNA degrading enzyme superfamily of proteins. It is postulated that its mechanism of action involves multiple interactions of angiogenin with other proteins through specific regions on the surface of the molecule. Potential angiogenin partners include heparin, plasminogen, elastase, angiostatin, actin, and the 170 kDa receptor on the surface of endothelial cells <Strydom, DJ
(1998) Cell. Mol. Life Sci. 54, 811-824>. Angiogenin is required for the angiogenic process. Tumor growth requires angiogenesis and therefore several anti-angiogenic agents have been produced and are currently in clinical trials. Patients with recurrent gastric cancer have also been shown to have higher angiogenin serum levels than patients with primary gastric cancer <Shimoyama, S. and Kaminishi, M. (2000) J.
Cancer Res. Clin. Oncol. 126, 468-474>. Therefore, angiogenin can be used as a diagnostic marker for cancer virulence assessment or an early marker of recurrence throughout follow-up. Angiogenin is a potent inducer of angiogenesis <Fett, JW; Strydo
m, DJ; Lobb, RR; Alderman, EM; Bethune, JL; Riordan, JF
And Vallee, BL (1985) Biochemistry 24, 5480-5486>. Angiogenesis
It is a complex process of angiogenesis and involves several distinct but interrelated steps at the cellular and biochemical level: (i) activation of endothelial cells by the action of angiogenic stimuli, (ii) activation. Adhesion and infiltration of endothelial cells to surrounding tissues,
And migration towards angiogenic stimuli, and (iii) formation of new microvasculature by endothelial cell proliferation and differentiation <Folkman, J. and Shing, Y. (1992) J. Biol.
Chem. 267, 10931-10934; Moscatelli, D. and Rifkin, DB (1988) Biochim.
Biophys. Acta 948, 67-85>.

Angiogenin binds to endothelial cells <Badet, J .; Soncin, F .; Guitton,
JD; Lamare, O .; Cartwright, T .; and Barritault, D. (1989) Proc. Natl
Acad. Sci USA 86, 8427-8431>, second messenger stimulation <Bicknell,
R. and Vallee, BL (1988) Proc. Natl. Acad. Sci. USA 85, 5961-5965
>, Mediation of cell adhesion <Soncin, F. (1992) Proc. Natl. Acad. Sci. USA 89,
2232-2236>, activation of cell-associated proteases <Hu, GF and Riordan,
JF (1993) Biochem. Biophys. Res. Commun. 197, 682-687>, induction of cell invasion <Hu, GF .; Riordan, JF; and Vallee, BL (1994) Proc. Na.
tl. Acad. Sci. USA 91, 12096-12100>, induction of endothelial cell proliferation <Hu, GF .;
Riordan, JF; and Vallee, BL (1997) Proc. Natl. Acad. Sci. US
A. 94, 2204-2209>, and the organization of tubular structure formation from cultured endothelial cells <Jimi,
SI .; Ito, KI .; Kohno, K .; Ono, M .; Kuwano, M .; Itagaki, Y .; and Isikawa, H. (1985) Biochem. Biophys. Res. Commun. 211, 476-483> Has been demonstrated to induce most individual events in the angiogenic process, including. Angiogenin also translocates the nucleus in endothelial cells via receptor-mediated endocytosis <Moroianu, J. and Riordan, JF (1994) P
Roc. Natl. Acad. Sci. USA 91, 1677-1681> and nuclear localization sequence-assisted nuclear import <Moroianu, J. and Riordan, JF (1994) Biochem. Biophys. Res. C.
ommun. 203, 1765-1772>. Since angiogenesis is a tightly controlled process under normal physiological conditions, abnormal angiogenesis has dire consequences leading to pathologies such as arthritis, diabetic retinopathy and tumor growth. It has now been demonstrated that the growth of virtually all solid tumors is angiogenesis-dependent <Folkman, J. (1989) J. Natl. Cancer Ins
t. 82, 4-6>. Angiogenesis also causes tumor cells to spread from the primary tumor,
Essential for metastasis development to provide a means to establish distant cell lines <Mahadeva
n, V. and Hart, IR (1990) Rev. Oncol. 3, 97-103; Blood, CH and Zetter BR (1990) Biochim. Biophys. Acta 1032, 89-118>. Therefore, interference with the tumor-induced angiogenic process could be an effective treatment for both primary and metastatic cancers.

It was first isolated from medium conditioned by human colon cancer cells (Fett et al. (1985),
Supra), and was subsequently shown to be produced by several other histological types of human tumors <Rybak, SM; Fett, JW; Yao, QZ .; and Vallee, BL (1987).
) Biochem. Biophys. Res, Commun 146, 1240-1248; Olson, KA; Fett, J.
W .; French, TC; Key, ME; and Vallee, BL (1995) Proc. Natl. Ac
ad. Sci. USA 92, 442-446>, angiogenin is also a component of human plasma and normally circulates at concentrations of 250-360 ng / ml <Shimoyama, S .; Gansauge,
F .; Gansauge, S .; Negri, G .; Oohara, T .; and Beger, HG (1996) Cancer
Res 56, 2703-2706; Blaser, J .; Triebl, S .; Kopp, C .; and Tschesche, H.
(1993) Eur. J. Clin. Chem. Clin. Biochem. 31, 513-516>. Several inhibitors of angiogenin function have been developed. These are (i) monoclonal antibody (mAb) <Fett, JW; Olson, KA; and Rybak, SM (1994).
Biochemistry 33, 5421-5427>, (ii) Angiogenin binding protein <Hu, G
-F .; Chang, SI .; Riordan, JF; and Vallee, BL (1991) Proc. Natl
Acad. Sci. USA 88, 2227-2231; Hu, GF .; Strydom, DJ; Fett, J. W.
.; Riordan, JF; and Vallee, BL (1993) Proc. Natl. Acad. Sci. U.
SA 90, 1217-1221; Moroianu, J .; Fett, JW; Riordan, JF; and V
allee, BL (1993) Proc. Natl. Acad. Sci. USA 90, 3815-3819>, (iii
) Placental RNA degrading enzyme inhibitor (PRI) <Shapiro, R. and Vallee, BL (198
7) Proc. Natl. Acad. Sci. USA 84, 2238-2241>, (iv) of angiogenin
Peptides synthesized based on C-terminal sequence <Rybak, SM; Auld, DS; St. Cl
air, DK; Yao, QZ .; and Fett, JW (1989) Biochem. Biophys. Res.
Commun. 162, 535-543>, and (v) inhibitory site-directed mutagenesis of angiogenin <Shapiro, R. and Vallee, BL (1989) Biochemistry 28, 7401-7.
408> is included. The present invention provides the protein / polypeptide of SEQ ID NO: 254. The invention also provides a bioactive fragment of SEQ ID NO: 254. In one embodiment, the polypeptide of SEQ ID NO: 254 is compatible with the corresponding polypeptide encoded by the human cDNA which is clone 106-006-1-0-E3-CS. A “bioactive fragment” is defined as a peptide or polypeptide fragment that has at least one of the biological functions of a full-length protein (eg, stimulation of angiogenesis). SEQ ID NO: 25
Compositions of four proteins / polypeptides or bioactive fragments thereof are also provided by the present invention. These compositions can be made according to methods known in the art.

The present invention also provides variants of the protein of SEQ ID NO: 254. These variants have at least about 80% of the amino acids encoded by SEQ ID NO: 254.
, Preferably at least about 90%, and most preferably at least about 95% amino acid sequence identity. Variants according to the invention also have at least one functional or structural characteristic of the protein of SEQ ID NO: 254. The present invention also provides bioactive fragments of variant proteins. Compositions of variants, or bioactive fragments thereof, are also provided by the present invention. These compositions can be made according to methods known in the art. Unless otherwise stated, the methods disclosed herein are carried out using the protein encoded by SEQ ID NO: 254, bioactive fragments of SEQ ID NO: 254, variants of SEQ ID NO: 254 and bioactive fragments of said variants. it can. Due to the redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequence of SEQ ID NO: 254. In a preferred embodiment, SEQ ID NO: 254 is clone 106-0.
Coded by 06-1-0-E3-CS. It is within the skill of one in the art to create these alternative DNA sequences that encode proteins having identical or substantially identical amino acid sequences. These mutant DNA sequences are therefore within the scope of the invention. As used herein, a "substantially identical" sequence means a sequence having amino acid substitutions, deletions, additions or insertions that does not substantially affect biological activity. Also included in this definition are fragments that retain one or more characteristic biological activities of the protein encoded by clone 106-006-1-0-E3-CS. A "recombinant nucleotide variant" is an alternative polynucleotide that encodes a particular protein. They can be synthesized, for example, by taking advantage of the "redundancy" of the genetic code. Diverse codon substitutions, such as specific restriction sites or silent changes that produce codon usage-specific mutations, optimize cloning into plasmid or viral vectors, or expression in specific prokaryotic or eukaryotic host systems, respectively Can be introduced to In one aspect of the invention SEQ ID NO: 254 and variants thereof can be used for polyclonal or monoclonal antibody production. The biologically active and immunogenic fragment of SEQ ID NO: 254, or variant proteins can be used for antibody production. Polyclonal and / or monoclonal antibodies can be produced by methods known to those of skill in the art. The antibodies produced in accordance with the present invention can be used in various detection assays known to those of skill in the art. The antibody can be used to agonize or antagonize the biological activity of the protein of sequence 254.

Sequence 254 can be used as a marker for individuals at risk of tumor development or recurrence. As shown in the above reference, angiogenin is found at a specific level in healthy individuals, usually at concentrations of 250-360 ng / ml. Therefore, a quantitative immunoassay can be used to detect abnormal levels of SEQ ID NO: 254, thereby identifying individuals at risk of developing tumors. Alternatively, the invention provides an antibody or fragment thereof specific for SEQ ID NO: 254, which is used in a routine immunoassay to screen the presence or absence of SEQ ID NO: 254. Alternatively, the nucleic acid encoding SEQ ID NO: 254, or a fragment thereof, can be used in a hybridization assay to detect and / or quantify the expression of SEQ ID NO: 254. Such hybridization assays are well known to those of skill in the art and can be performed on a variety of samples, including but not limited to tumor cells, biopsy tissue, or normal tissue. Molecules that functionally suppress the action of angiogenin (Strydom, DJ, (1998) Cel
l. Mol. Life Sci. 54, 811-824) can be used to treat tumor patients. Because angiogenin is required for tumor angiogenesis, molecules that inhibit angiogenin bioactivity can be used to reduce tumor angiogenesis and control tumor growth. Accordingly, another aspect of the invention provides a molecule that suppresses or reduces the biological activity of SEQ ID NO: 254. One embodiment provides a neutralizing antibody to suppress the biological activity of SEQ ID NO: 254. These neutralizing antibodies have been chimerized or humanized according to methods known in the art to minimize the immunogenicity of the molecule for use in patients. Neutralizing antibodies can be used in combination with other known therapeutic modalities for tumor therapy. Another embodiment of the invention utilizes the concept that the expression of a particular gene can be suppressed by an oligonucleotide having a nucleotide sequence complementary to the mRNA transcript of the target gene. This suppression is caused by selectively hindered translation and is referred to as "antisense" methodology. In addition, the "antigene" or "triplex" methodology also uses oligonucleotides complementary to selective sites in double-stranded DNA to form triple-stranded complexes that selectively repress gene transcription. By doing so, the expression of the gene can be suppressed. “Antisense” and “antigene” methodologies can be used to suppress or reduce gene expression of SEQ ID NO: 254, thereby providing therapeutic benefit to the patient being treated. Treatments of individuals using antigene and antisense methodologies are known to those of skill in the art (eg, "Antisense Therapeutics") Agrawal, S. (ed), Human.
a Press, 1996; Crooke, ST, and Bennett, CF (1996) Annu. Rev. Phar
macol. Toxicol. 36, 107-129; "Prospects for the Therapeutic Use of Antigene Oligonucleotid
es ")", Maher, LJ (1996) Cancer Investigation 14 (1), 66-82. The full text of each is incorporated herein by reference).

As an additional example, US Pat. No. 5,098,890 relates to antisense oligonucleotides complementary to the c-myb oncogene and antisense oligonucleotide therapeutics for certain cancerous conditions. US Patent 5,135,917 describes human interleukin-
1 Supplies antisense oligonucleotides that suppress receptor expression. US Patent 5
, 087,617 provide a method for treating cancer patients with antisense oligonucleotides. US Pat. No. 5,166,195 provides oligonucleotide inhibitors of HIV. US Pat. No. 5,004,810 provides oligomers that can hybridize with herpes simplex virus Vmw65 mRNA and suppress replication. US Patent 5,194,42
8 supplies antisense oligonucleotides with antiviral activity against influenza virus. US Pat. No. 4,806,463 provides antisense oligonucleotides and methods of using them to suppress HTLV-III replication. US Pat. No. 5,286,717 relates to phosphorothionate, a mixed-chain oligonucleotide complementary to oncogenes. US Pat. Nos. 5,276,019 and 5,264,423 relate to phosphorothionate oligonucleotide analogs used to prevent replication of exogenous nucleic acids in cells. The entire text of each of these patents is incorporated herein by reference. The invention also provides a modified / derivatized nucleic acid encoding SEQ ID NO: 254. These include modifications that increase the stability / affinity of these targeted compounds. DN
Phosphorothionate analogs of oligodeoxynucleotides (ODNs), in which the non-bridging phosphoryl oxygen in the backbone of A is replaced by sulfur (<S> ODNs), are essentially their unaltered phosphodiester counterparts. Is more stable than. Other derivatives, such as those where the sugar oxygen group is alkylated, show enhanced target affinity. <S>
ODNs have excellent biological activity, pharmacology, pharmacokinetics and safety in vivo (Agrawal (1996), supra). Successful repression of a particular gene function involves diversifying sites on a particular mRNA sequence, including AUG translation initiation codon, 5'transcription initiation site, 3'stop codon and sequences in the 5'and 3'untranslated regions. Achieved by targeting. These derivatized nucleic acids can be used in any of the above methodologies. [Protein of SEQ ID NO: 387 (Internal Designation Number 105-073-2-0-A7-CS)] The protein of SEQ ID NO: 387 encoded by the cDNA of SEQ ID NO: 146 is liver,
It is expressed in the ovary and prostate and overexpressed in the salivary glands. The protein of SEQ ID NO: 387 is characterized by an alpha / beta hydrase fold, which belongs to the abhydrase family and is common to several hydrolases with widely differing phylogenetic origins and catalytic functions (Protein Eng 1992; 5: 197-211, the entire disclosure of which is incorporated herein by reference).

The core of each enzyme is an alpha / beta sheet (a sheet rather than a barrel) that contains the eight chains bound by the helix. This enzyme diverges from its common ancestor and is thought to conserve the sequence of catalytic residues. All are on loops and have a catalytic tri-fold axis consisting of elements that are the most conserved structural feature of the folded structure. Epoxide hydrolases are a family of enzymes that hydrolyze various foreign and endogenous epoxides to their corresponding diols. Epoxide hydrolase
Water is added to the epoxide to form the corresponding diol. Based on sequence similarity, mammalian soluble epoxide hydrolases are two evolutionarily distinct domains, an N-terminal domain similar to bacterial haloacid dehalogenases, soluble plant epoxide hydrolases, and microsomal epoxides. Contains a hydrolase, a C-terminal domain similar to bacterial haloalkane dehalogenases (DNA Cell Biol 14: 61-7
1 (1995), the entire disclosure of which is incorporated herein by reference. Human epoxide hydrolase adds water to epoxide,
Form the corresponding dihydrodiol. Enzymatic hydration is essentially irreversible, producing predominantly less reactive metabolites that can be conjugated and excreted.
The reaction of epoxide hydrolase is therefore generally regarded as detoxification. Generally, the function of epoxide hydrolase is ultimately accompanied by the excretion of diol. However, reactivation of certain diols may occur due to secondary epoxidation. Epoxide hydrolases also inactivate epoxides present during metabolism of endogenous compounds. Lipophilic xenobiotics tend to accumulate in tissues and must be converted to water-soluble compounds in order to be excreted. In this conversion process, reactive intermediates are produced. When biotransformation fails to detoxify these reactive intermediates, they can either covalently react with key targets such as genetic material or initiate harmful reaction chains such as lipid peroxidation. I have something to do. Therefore, epoxide hydrolase is believed to be responsible for carcinogenic and mutagenic phenomena (Exp Pathol 1990; 39 (3-4): 195-6.). Furthermore, the interaction between epoxide hydrolase activity and alcohol metabolizing enzyme is
It suggests that it may be associated with susceptibility to alcohol-dependent liver disease and hepatocellular carcinoma (Toxicol. Lett. 10; 115 (1): 17-22 (2000), the disclosure of which is incorporated herein by reference in its entirety. Have been incorporated). Compounds containing epoxide functionality have become common environmental pollutants because of their wide use as insecticides, disinfectants and industrial precursors. Such compounds also occur as a result of spontaneous oxidation of products, by-products or intermediates in normal metabolism, and membrane lipids (Br
Ash et al., Proc. Natl. Acad. Sci., 85: 3382-3386 (1988), and Sevanian, A.
Et al., Molecular Basis of Environmental Toxicology (Bhatnager, RS, ed.)
pp. 213-228, Ann Algor Science, Michigan (1980)). As a three-component cyclic ether, epoxides are often found to be extremely reactive, yet cytotoxic, mutagenic and carcinogenic (Sugiyama, S. et al., Li.
fe Sci 40: 225-231 (1987)). Cleavage of ether bonds in the presence of electrophiles often results in adduct formation. As a result, epoxides participate as a proximate toxin or mutagen for many xenobiotics. The detoxification reaction using an epoxide hydrolase generally reduces the hydrophobicity of the compound, resulting in a stronger polarity and excretable substance.

The protein of SEQ ID NO: 387 or part thereof is considered to be a hydrolase, preferably an epoxide hydrolase. Preferred polypeptides of the invention are 2
~ 132, 52-137, 29-120, 12-137, 19-136, 151-209, 30-108, and 35-
A polypeptide comprising the amino acid sequence of SEQ ID NO: 387 from position 108. Other preferred polypeptides of the present invention are fragments of SEQ ID NO: 387 that include any of the biological activities described herein. The hydrolytic activity of the proteins of the invention or parts thereof is described in Cancer res, the entire disclosure of which is incorporated herein by reference.
40 (7): 2552-6 (1980); Exp Pathol 39 (3-4): 195-6 (1990) can be used for assay using any of the known assay methods known to those skilled in the art. The present invention provides a protein of the invention for the diagnosis, prevention and / or treatment of several disorders associated with overexpression of the protein of the invention, including alcohol-dependent liver disease, hepatocellular carcinoma, ovarian and prostate cancer. Or methods and compositions using portions thereof. In addition, the proteins of the invention, or portions thereof, can be used to identify inhibitors for mechanistic and clinical applications. Such inhibitors then identify or quantify the protein of the invention in the sample, as well as amyloidosis, colitis, lysosomal disorders, arthritis, muscular dystrophy, inflammation, tumor invasion, glomerulonephritis, parasitic infections, Diagnosis of disorders in which the hydrolytic activity of the protein is undesirable and / or detrimental, such as, but not limited to, Alzheimer's disease, periodontal disease, and cancer metastasis, such as disorders characterized by tissue degeneration. ,
Can be used for treatment or prevention. In another embodiment, the invention relates to methods and compositions using the proteins of the invention or portions thereof as marker proteins for selectively identifying tissues, preferably ovaries, liver or prostate, more preferably salivary glands. . For example, the proteins of the invention or portions thereof can be used to synthesize specific antibodies using techniques known to those of skill in the art. Such tissue-specific antibodies are then used in forensic samples,
It can be used to identify tissues of unknown origin, such as differentiated tumor tissues that have metastasized to different body sites, etc., or to classify different tissue types in tissue cross sections using immunochemistry.

[Protein of SEQ ID NO: 398 (Internal Designation No. 160-31-3-0-E4-CS)] The protein of SEQ ID NO: 398 encoded by the cDNA of SEQ ID NO: 157 is overexpressed in fetal brain and is diversified. Shows homology with various hydrolases. The proteins of the invention also show a motif characteristic of the isochorismate acid degrading protein from positions 17-147. Furthermore, the proteins of the invention are otherwise spliced forms of the anonymous human protein. The protein of SEQ ID NO: 398 or a part thereof is considered to be a hydrolase, preferably acting on an ether bond, and more preferably an ether hydrolase. A preferred polypeptide of the invention is a polypeptide comprising the amino acids of sequence 398 from positions 17-147. Other preferred polypeptides of the present invention are fragments of sequence 398 that have any of the biological activities described herein. The hydrolytic activity of the protein of the present invention or a part thereof is reported in US Patent Nos. 5,445,942, 5,445,9.
56, 6,017,746 and 5,871,616, and Rusnak et al., 1990; Biochemistry 2
9 1425-1435 can be assayed using any of the assay methods known to those of skill in the art, including those described. In another embodiment, the invention provides a protein of the invention, or a portion thereof, to tissue,
Preferably it relates to methods and compositions for use as a marker protein for selectively identifying fetal brain. For example, the proteins of the invention, or portions thereof, can be used to synthesize specific antibodies using techniques known to those of skill in the art, including those described herein. Such tissue-specific antibodies can then be used to identify tissues of unknown origin, such as differentiated tumor tissues that have metastasized to forensic samples, different body sites, etc., or use immunochemistry to identify different tissues in a tissue cross section. It can be used to classify types. [Proteins of SEQ ID NOs: 260 and 265 (internal designation numbers 116-004-3-0-A6-CS and 116-091-1-0-D9-CS)] Encoded by the cDNA of SEQ ID NO: 19 in the liver and testis The overexpressed protein of SEQ ID NO: 260 is an isoform of the protein of SEQ ID NO: 265, which is encoded by the cDNA of SEQ ID NO: 24 overexpressed in liver. Both proteins are mouse EPCS26 (Hemberger M. et al. Dev., Having Genbank accession number AF250838.
Biol. 222, 158-169 (2000)). The proteins of SEQ ID NOs: 260 and 265 contain a signal peptide (cleavage site at position 18) that allows the export of the protein to the extracellular domain, the export to the cell membrane, or specific subcellular localization. The cDNA encoding EPCS26 has been shown to be specifically expressed during the process of trophoblast infiltration.

Implantation and placenta formation are major processes in mammalian embryonic development. They physically connect the embryo to its mother and are essential for adequate nutrient and gas exchange. The extraembryonic cell line is the first to differentiate in developmental conceptus, reflecting the importance of this cell for the establishment of fetal-maternal connections. During mouse development, the outer layer of the blastocyst, the mural vegetative ectoderm, was formed on day 5 of pregnancy (e5).
Initiate differentiation into primary trophoblast giant cells. These cells invade the uterine epithelium and penetrate deep into the stroma. At the same time, polar trophectoderm cells continue to proliferate to form trophoblast cones. At e7 (7th day of gestation), cells outside the trophoblast cones begin to differentiate into secondary trophoblast giant cells. Infiltration of the uterine stroma by these cells is essential for successful placentation (Cross et al. Science 26
6, 1508-1518 (1994)). Trophoblast infiltration triggers the secretion of proteinases, which degrade extracellular matrix molecules. Mouse trophoblasts have been shown to synthesize and secrete serine proteases, matrix metalloproteinases, and cysteine proteinases. Trophoblast infiltration is a highly controlled process. The decidua limits invasion by secreting proteinase inhibitors. Proteinases and proteinase inhibitors have antagonistic functions in implantation and placentation that can be reflected by the interrelationship of their expression patterns (Alexander et al., Development 122, 1723-1736 (1996).
). During tumor invasion and metastasis, basement membrane degradation is often achieved by a proteinase involved in implantation and normal trophoblast invasion (Strickland and Richa
rds Cell 71, 355-357 (1992), Wilson et al., Proc. Natl. Acad. Sci. USA 94.
, 1402-1407 (1997)). Uncontrolled invasion of trophoblasts, as in choriocarcinoma, results in one of the best known metastatic tumors (Strickland
and Richards Cell 71, 355-357 (1992)). The lack of function of the proteins of the invention results in invasion of uncontrolled trophoblasts and one of the most well known metastatic tumors, such as in the case of choriocarcinoma (Strickland and Richards Cell 71, 355-357 (1992)).

The proteins of SEQ ID NOs: 260 and 265 or parts thereof, preferably during embryogenesis,
More preferably, it is believed to be involved in protein degradation during trophoblast infiltration. The protein of the present invention or a part thereof may act as a secretory proteinase that decomposes extracellular matrix molecules, or conversely, as a proteinase inhibitor. A preferred polypeptide of the invention is a polypeptide comprising the amino acids of SEQ ID NO: 260 from positions 7-122 and the amino acids of sequence 265 from positions 7-81. Other preferred polypeptides of the present invention are fragments of SEQ ID NOs: 260 and 265, which have any of the biological activities described herein. The proteolytic activity of the proteins of the invention or portions thereof can be assayed using any of the assays known to those of skill in the art, including those described in US Patents 6,069,229 and 5,861,267. The protease inhibitor activity of the protein of the present invention or a part thereof can be assayed using any of the assays known to those skilled in the art, as well as methods for measuring inhibition constants known to those skilled in the art (Fersht, ENZYME STRUCTURE AND MECHANISM, 2nd ed., WH Free
See man and Co., New York, (1985)). Furthermore, the protein of the present invention or a part thereof is amyloidosis, colitis, lysosomal disease, arthritis, muscular dystrophy, inflammation, tumor infiltration, glomerulonephritis, parasitic infection, Alzheimer's disease, periodontal disease, cancer. Including metastases and choriocarcinoma,
It can be used for the diagnosis, treatment or prevention of disorders characterized by unwanted and / or harmful hydrolytic activity, such as, but not limited to, disorders characterized by tissue degeneration. For diagnostic purposes, expression of the proteins of the invention can be studied using Northern blot, RT-PCR, immunoblotting methods described herein and compared to expression in control individuals. Alternatively, inhibitors of protein activity can be developed and used to inhibit and / or reduce its activity using any of the methods known to those of skill in the art. Overexpression of the proteins of the invention or portions thereof can be achieved using any of the gene therapy methods described herein. In another embodiment, the invention provides a protein of the invention, or a portion thereof, to tissue,
Preferably, the protein of SEQ ID NO: 260 relates to liver and testis, preferably the protein of SEQ ID NO: 265 relates to methods and compositions used as a marker protein for selectively identifying liver. For example, the proteins of the invention, or portions thereof, can be used to synthesize specific antibodies using techniques known to those of skill in the art, including those described herein. Such tissue-specific antibodies can then identify tissue of unknown origin, such as differentiated tumor tissue that has metastasized to forensic samples, different body sites, etc., or use immunochemistry to identify different tissue types in tissue cross-sections. Can be used to classify.

[Protein of SEQ ID NO: 265 (Internal Designation No. 116-088-4-0-A9-CS)] The protein of SEQ ID NO: 265 encoded by the cDNA of SEQ ID NO: 24 is overexpressed in testis and liver. . This protein of the invention is a CdX protein (Toniolo et al., Proc Natl, also referred to as UBL4, both found in humans.
Acad Sci USA 1988; 85: 851-5) (GENPEPT accession number L44140), and mouse species (GENPEPT accession number J04761). In addition, the protein of SEQ ID NO: 265, which is 174 amino acids long, is similar in size to the ubiquitin-like proteins and exhibits the pfam consensus domain from position 1-82, which is characteristic of ubiquitin family proteins. Ubiquitin is a 76-amino acid protein that is present in all eukaryotic cells and is extremely well conserved from protozoa to vertebrates (Jentsch et al.
Trends Cell Biol 2000; 10: 335-42). It is involved in various cellular processes such as ATP-dependent selective degradation of cellular proteins, maintenance of chromatin structure, regulation of gene expression, stress response, ribosome biosynthesis, cell cycle progression, signal transduction, transcription and antigen presentation. Play a role (Wilkinson et al., Annu Rev Nutr 1995; 15
: 161-89). The first ubiquitin is covalently bound to the target protein via an isopeptide bond between the C-terminal glycine residue of ubiquitin and the lysine residue of the substrate (-amino group). Efficient proteasome target signal generation In order to do so, other ubiquitins bind to the first ubiquitin via an isopeptide bond to form a branched polyubiquitin complex (Thrower et al., EMBO J 2000; 19: 94-102).
Covalent attachment of ubiquitin to a protein labels the protein for subsequent degradation by a multi-component enzyme complex known as the 26S proteasome. Genes encoding ubiquitin-like proteins fall into two separate classes (Hershko et al., Annu Rev Biochem 1992; 61: 761-807). Proteins belonging to the first class are frequently designed as ubiquitin-like modifiers or UBLs. They produce polyubiquitin molecules that consist of precise head-to-tail repeats of ubiquitin, with variable number of repeats. These linear polymers of ubiquitin are covalently linked by a peptide bond between the C-terminal glycine and N-terminal lysine residues of adjacent ubiquitin molecules. The second class of proteins is customarily named ubiquitin domain proteins, or UDP. These proteins carry one domain, the N-terminal domain associated with ubiquitin fused to a C-terminal ribosomal domain of 52 or 76-80 amino acid residues (Finley et al., Nature 1989; 338: 394. -401). These proteins are not conjugated to other proteins and function as a heterogeneous group of proteins. To date, this family has RAD23, DSK2, PLIC-1, PLIC-2 / Chap1, XDRP1
, BAG-1, BAT3 / Chap2, Scythe, Parkin, UIP28, UBP6, Elongin B and
Including GdX. Furthermore, the protein of the present invention, SEQ ID NO: 265, clearly belongs to the UDP family because it exhibits a single ubiquitin N-terminal common domain, which is characteristic of this protein family subset.

[0167] UDP is associated with the catalytically active 26S proteasome and RAD23 (Schauber et al., Nature 199).
8; 391: 715-8), hPLIC-1 and hPLIC-2 (Kleijnen et al., Mol Cell 2000; 6:40).
9-19), and BAG-1 (Luders et al., J Biol Chem 2000; 275: 4613-7), removal of ubiquitin from conjugates with UBP6 (Wyndham et al., Protein Sci 1997; 8:
1268-75) as well as negative regulation of multi-ubiquitin chain assembly of RAD23 (Ortolan et al., Nature cell Biol 2000; 2: 601-8) and is involved in the regulation of proteolysis. In addition, there has been an increase in empirical evidence from studies showing that UDP has protein folding (Luders et al., J Biol Chem 2000; 275: 4613-7), apoptosis (Kaye et al., FEBS Lett 2000; 467: 348-55) and nucleotide excision repair ( de L
aat et al., Genes Dev 1999; 13: 768-785). UDP family proteins are associated with RAD23-induced xeroderma pigmentosum (Masutani
Et al., EMBO J 1994; 13: 1831-43), and parkin's disease by Parkin (Kitad
et al., Nature 1998; 392: 605-8) and has been shown to be directly associated with the etiology of several diseases. Furthermore, accumulation of ubiquitin-like proteins or proteins due to aberrant ubiquitin binding has been found in multiple human disorders. Most, but not all, of Alzheimer's disease (van Leeuwen et al., Scien
ce 1998; 279: 242-7), diffuse Lewy body disease (Iseki et al., J Neurol Sci 1997; 14).
6: 53-7), Huntington's chorea (Scherzinger et al., Cell 1997; 90: 549-58),
And in the central nervous system such as amyotrophic lateral sclerosis (Leigh et al. Brain 1991; 114: 775-88). In most disorders, ubiquitin-binding proteins accumulate intracellularly, forming aggregates called inclusion bodies that have a characteristic appearance on histological examination. In addition, the abnormal accumulation of ubiquitin-binding proteins was reported by Von-Hippel Linda.
u disease (Kamura et al., Proc Natl Acad Sci. USA 2000; 97: 10430-5) and liver of patients with alcohol dependent hepatitis (Ohta et al., Lab Invest. 1988; 59: 848-56). Hepatocyte components are released into the circulation of alcohol-dependent hepatitis (
Sorbi et al., Am J Gastroenterol 1999; 94: 1018-22). It is believed that the protein of SEQ ID NO: 265, or a portion thereof, is preferably involved as a ubiquitin-like protein, more preferably as a ubiquitin domain protein, in controlling proteolysis. Furthermore, the protein of the present invention is
It may be involved in protein folding, apoptosis, and nucleotide excision repair. A preferred polypeptide of the invention is a polypeptide comprising the amino acids of SEQ ID NO: 265 from positions 1-82. Other preferred polypeptides of the present invention are fragments of SEQ ID NO: 265 having any of the biological activities described herein.

In an embodiment, the invention relates to compositions and methods that employ a protein of the invention or a portion thereof to remove, identify, or suppress contaminating proteases in a sample. A composition comprising a polypeptide of the invention can be added to a biological sample as a "cocktail" with other protease inhibitors to prevent degradation of the protein sample. The advantage of using a cocktail of protease inhibitors is that they can inhibit a wide range of proteases without necessarily knowing the specificity of the protease. The use of a cocktail of protease inhibitors also protects samples from a wide range of unknown proteases, which can contaminate protein samples from vast numbers of sources. Such protease inhibitor cocktails (eg Si
Commercially available cocktails from gma) are widely used in research laboratory assays to inhibit proteases that are sensitive to the degradation of the protein of interest for which the assay is being performed. For example, a protein of the invention or portion thereof is added to a sample where proteolytic degradation by contaminating proteases is undesirable. Alternatively, the protein of the invention, or a portion thereof, is bound to a chromatographic support, using techniques known in the art, alone or in combination with other protease inhibitors, to make affinity chromatography columns. May be. Samples containing unwanted proteases are stripped of proteases through a column. Alternatively, similar methods can be used to identify new proteases. Another embodiment of the invention is a composition using a protein of the invention or a portion thereof to develop an assay for immunohistochemical detection of testicular malignant tissue in which the protein of the invention is overexpressed and Regarding the method. For example, this is Nazeer et al., Onc
ol Rep (1998); 5: 1425-9, can be used for staging of testicular cancer-disseminated lymph nodes using techniques and methods described in detail. The ability to specifically visualize malignant tissue (and cells derived from said tissue) has been shown to be specific for determining the origin, for example to identify cancer cells, and for example for histological slide evaluation. It is useful for numerous applications, including facilitating identification of cells and tissues. In another embodiment, the invention provides xeroderma pigmentosum, Von-Hippel Lindau disease, alcohol-dependent hepatitis, Alzheimer's disease, diffuse Lewy body disease, Huntington's chorea,
And a composition using the protein of SEQ ID NO: 265 or a portion thereof to diagnose, treat, and / or prevent disorders, including but not limited to neurodegenerative diseases such as amyotrophic lateral sclerosis. Or regarding the method. Detection of polyubiquitin-binding protein conjugates in biological samples, such as brain tissue for diagnosis of neurodegenerative disorders, or liver and serum or plasma for diagnosis of alcohol-dependent hepatitis,
Immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), or Northern blot, RT-PCR, immunoblot as described herein, and Mimnaugh et al., Electro.
Other techniques known to those of skill in the art, including those described in phoresis 1999; 20: 418-28, can be used with antibodies or nucleic acids capable of detecting expression of the proteins of the invention. Expression of the proteins of the invention in patient samples is then compared to expression in control individuals.

In yet another embodiment, the present invention provides a pigmented dry skin using the protein of the present invention, a part thereof, or another compound developed by using the protein as a nucleic acid, an antibody, or a chemical substance. Disorders, including, but not limited to, neuropathy, Von-Hippel Lindau disease, alcohol-dependent hepatitis, Alzheimer's disease, diffuse Lewy body disease, Huntington's chorea, and neurodegenerative diseases such as amyotrophic lateral sclerosis. Treat,
Compositions or methods for attenuating and / or preventing. In a preferred embodiment, a protein or other compound that targets the invention or a portion thereof has a ubiquitin-like protein or protein accumulation due to aberrant ubiquitin binding that has been discovered and is associated with disorders such as those involved in the pathogenesis of disease. It can be used for treatment, prevention and / or attenuation. For example, a protein or other compound that targets the protein of SEQ ID NO: 265 can be administered to treat or reduce the symptoms of a patient suffering from Alzheimer's disease or other neurodegenerative disorders. [Protein of SEQ ID NO: 408 (Internal Designation No. 174-8-2-0-C10-CS)] The protein of SEQ ID NO: 408, which is found in the salivary glands and brain and is encoded by the cDNA of SEQ ID NO: 167, is transmembrane. Is homologous to Drosophila melanogaster protein (STR: Q9V641). SEQ ID NO: 408, which is 345 amino acids long
Protein exhibits a rhomboid pfam domain from positions 186-323, 101-121,167
It is predicted to have 6 transmembrane domains from positions 187, 204-224, 243-263, 273-293, 298-318. Rhombic genes have been identified in flies and in diverse organisms such as Arabidopsis, yeast, bacteria and mammals. Diamond-shaped human and rat homologues have been identified (Pascal et al., 1998; FEBBS Lett. 429; 337-340). This extremely widespread conservation means that rhomboid family proteins have basic functions in many cells. The Drosophila rhombus has 6 transmembrane domains and an amino-terminal hydrophobic region like the proteins of the invention. The 355 amino acid long Drosophila rhomboid protein is known to control multiple aspects of fly development, and especially to establish positions along the dorsoventral axis and then again later to identify the fate of neuronal progenitor cells. . Diamond expression is sufficient to activate EGF receptor (EGFr) signaling in all tissues of Drosophila, and deletion of diamonds reduces EGFr signaling in almost all tissues (
Or deletion) (Guichard et al .: 1999 Development; 126, 2663-2676).
). In mammals, the Drosophila EGF receptor regulates many aspects of growth and development. Three activating ligands of Drosophila EGFr have been described, the most important developmentally being the TGFα-like molecule Spitz (Rutledge et al., 1992).
Genes & Dev. 6; 1503-1517). None of the rhombus-like proteins from species other than Drosophila have a well-specified function. However, the diamond has a major role in intracellular signaling by functioning as an activator of the EGF receptor and possibly controlling activation of Spitz, a TGF-like ligand,
There is firm genetic evidence from Drosophila (Guichard et al .: 1999 De
velopment; 126, 2663-2676). Indeed, rhomboid expression is the major rate-limiting step in activation of the Ras / MAP kinase pathway by EGFr.

Like mammalian TGFα, Spitz is synthesized as a functionally inactive transmembrane protein; the proteolytic release of the extracellular portion of subsequent molecules produces soluble and potent EGFr ligands (Golembo et al. , 1996; Development; 122;
3363-3370). Unlike all other essential components of EGFr signaling, diamond expression is strictly restricted to the site of signaling activity. It has been proposed that diamonds achieve a major role in the pathway by controlling the proteolytic cleavage of Spitz (Wasserman et al .: 2000; Genes &development;
14; 1651-1663). The excellence in the diamond-shaped pathway, which is essential for developmental and growth regulation as well as the EGFr / Ras / Map kinase cascade, provides a strong incentive to understand its molecular mechanism. By analogy with a similarly processed mammalian EGFr ligand, Spitz cleavage is predicted to be catalyzed by an ADAM-like protease (Bla.
ck et al., 1998; Curr. Opin. Cell. Biol. 10; 654-659), but the diamonds do not resemble known proteases. The Drosophila eye serves as a useful model for studying the mechanisms of EGFr and Ras signaling. Different roles for at least five receptors have been identified (see Wasserman et al .: 2000; Genes &development; 1
4: 1651-1663), the most characteristic is the function of gradually adding cells to generate individual eyes, which are individual units of the compound eye of the fly. Each eye contains 8 photoreceptors, 4 pyramidal cells secreting lens material, and an average of 8 pigment cells. Fly EGFr
Has been shown to play a role in controlling cell survival in the developing eye (Do
minguez et al., 1998; Curr. Biol. 8; 1039-1048). The EGFr signaling pathway is conserved between flies and vertebrates. The EGFr family consists of four members, HER1 (c-erbB1, EGFR), which are expressed in a wide range of cells.
HER2 (c-erbB2), HER3 (c-erbB3), HER4 (c-erbB4) (Gullick WJ 1
998; Br. Cancer Res: Treat .; 52, 43-53). TGFα and its homologues have been found to be the most abundant ligands of the EGF / TGFα receptor in most parts of the brain (Kaser et al. (1992) Brain Res: Mol Brain Res: 16: 316-322.
). In contrast to EGF, which is only present in smaller discrete areas, TGFα appears to be widely distributed in various regions of the brain, suggesting that TGFα plays a physiological role in brain tissue. These multiple receptor sites for TGFα in the brain
We suggest that GF has important utility in promoting the differentiation and function of normal brain cells.

Transforming growth factor alpha (TGFα) is a relative of epidermal growth factor (EGF) and, like EGF, affects cells by binding EGF receptors. The exact physiological role of TGFα has not yet been elucidated, but it appears to be important in the development of the eye and hair follicles and may also be involved in both the immune system and wound healing. (Kumar et al .; 1995 Cell Biology International, 19: 5, 37.
See 3-388). The EGF family of receptors currently contains four EGF receptors. EGFR2
The receptor is also referred to as ERB-2, a molecule that is useful for a variety of diagnostic and therapeutic effects (Prigent, SA and Lemoine, NR, (1992) Pro
g Growth Factor Res, 4: 1-24). TGFα appears to be a ligand for one or more of these receptors as well as an as yet unidentified new EGF-type receptor. T
The use of GFα can aid in the identification, characterization and cloning of such receptors. For example, the EGF receptor gene represents a cell homolog of the v-erb-B oncogene of avian erythroblastosis virus. Overexpression of the EGF receptor or loss of the kinase regulatory segment of the protein results in neoplastic transformation of cells (Manjusri, D. et al., (19
91) Human cytokines, 364 and 381). The ErbB family of EGF receptors and related tyrosine kinase receptors are actually involved in human cancer. It is generally believed that hyperactive receptor signaling promotes dysregulation of growth regulation, is involved in the initiation of malignant transformation, and disrupts developmental programs. However, little is known about ErbB physiological regulation in humans. Drosophila, Drosophila melanogaster, 4 ErbB
It has one receptor that is homologous to the receptor. These experimental results in flies are relevant for human receptor studies in development and disease, as the signaling mechanism is well conserved between flies and mammals. Two areas of recent progress are highlighted. First, several signal modulators have been identified, including three EGF receptor inhibitors, some of which are human homologs. Second, we identify that signaling molecules integrate into the regulatory network, which produces the activation profile required for development (positive and negative feedback regulation of EGF receptor signaling is a central theme. Has become). Because of the substantial clinical importance of the EGFr pathway, it is therefore possible to discover whether rhomboid-like proteins also have functions in other higher organisms, including mammals, similar to those observed in Drosophila. is important.

The protein of SEQ ID NO: 408, or a portion thereof, is believed to be involved in the regulation of cell signaling. Preferably, the protein of the invention, or a portion thereof, is likely through the control of activation of EGFr ligands such as EGF, TGFα and TGFα-like factors, and more likely through proteolytic cleavage of such ligands. And is involved in the activation of cell signaling mediated by EGFr. Preferred polypeptides of the present invention are 186-323, 101-121, 167-187, 204-224, 243-263, 273-293,
And a polypeptide comprising the amino acids of SEQ ID NO: 408 from positions 298-318.
Other preferred polypeptides of the present invention are fragments of SEQ ID NO: 408 that include any of the biological activities described herein. The proteolytic activity of a protein of the invention or a portion thereof, as well as its involvement in the regulation of cell signaling by activation of EGFr, can be assayed using any of the assays known to those of skill in the art. Embodiments of the present invention include a protein of the present invention or a portion thereof for identifying and / or quantifying EGF receptor, preferably vertebrate EGF receptor, and more preferably human ErbB receptor activation in a biological sample. Compositions and methods using, and therefore are used in assay methods and diagnostic kits for the quantification of such activation in body fluids, tissue samples, and in mammalian cell culture. Assessment of EGF receptor activation can be performed using any of the assays known to those of skill in the art. Preferably, a defined amount of the protein of the present invention or a part thereof is added to the sample under conditions that allow activation of EGFr. EGFr activation is then assayed and finally compared to controls using any of the techniques known to those of skill in the art. The invention also relates to a diagnostic assay for detecting the level of modification of a protein of the invention in various tissues, wherein the overexpression of the protein compared to normal control tissue samples results in abnormal growth. This is because it is possible to detect the presence of specific disease states such as skin disorders, eye disorders and inflammation. Assays used to detect levels of a polypeptide of the invention in a sample from a host are known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western Blot assays and Preferably it includes an ELISA assay. The present invention also relates to the use of SEQ ID NO: 167 or its complement as a diagnostic tool. Detection of mutant forms of the nucleotide sequence of SEQ ID NO: 167 of the present invention may be performed, for example, by improper wound healing, improper neural function, ocular disorders, kidney and liver disorders, hair follicle development, angiogenesis and embryogenesis. It is possible to diagnose diseases or susceptibility to diseases caused by underexpression of the polypeptide of the present invention. SEQ ID NO: 167 of the present invention
Individuals responsible for mutations in the human nucleotide sequence of
It can be detected at the DNA level. Diagnostic nucleic acids are obtained from patient cells such as blood, urine, saliva, biopsy and autopsy materials. Genomic DNA may be used directly for detection or may be enzymatically amplified using PCR prior to assay (Saiki et al.,
(1986) Nature, 324: 163-166). RNA or cDNA can also be used for similar purposes. As an example, PCR primers complementary to a nucleic acid encoding a polypeptide of the invention can be used to identify and analyze that mutation. For example, deletions and insertions
It can be detected by changing the size of the amplification product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA, or alternatively radiolabeled antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by digestion with RNAse A or differences in melting temperatures.

In other embodiments, the proteins of the invention, or portions thereof, can be administered to cancer and hyperaldosterone, hypoadrenocortical (hypoadrenocortical) using any of the methods and / or techniques described herein. Addison's disease), hyperthyroidism (Graves' disease), hypothyroidism, colorectal polyps, gastritis, gastric and duodenal ulcers, ulcerative colitis, and other associated abnormal cell differentiation, proliferation or degeneration, including Crohn's disease. It can be used for the diagnosis, treatment and / or prevention of disorders, disorders associated with dysregulation of growth regulation, such as neurodegenerative disorders such as Parkinson's disease or Alzheimer's disease. For diagnostic purposes, expression of the proteins of the invention can be studied using Northern blot, RT-PCR, immunoblotting methods described herein and compared to expression in control individuals. Furthermore, the proteins of the invention or parts thereof can be used for the evaluation of disease progression and efficacy of clinical treatment. Suppression of the expression of a protein of the invention or a portion thereof to suppress EGFr activation includes numerous means known to those of skill in the art, including those described in this application such as antisense nucleotides or triple helix strategies. Can be achieved by [Protein of SEQ ID NO: 291 (internal designation number 180-19-4-0-F4-CS)] The protein of SEQ ID NO: 291 encoded by the cDNA of SEQ ID NO: 50 is a tissue inhibitor of the metalloproteinase (TIMP) family. It is homologous to protein. The protein of the present invention (207 amino acids) is a metalloproteinase inhibitor 1 precursor (TIMP-1, 207 amino acids) human protein (SwissProt P01033).
Are highly homologous to each other and are likely to be variants thereof. The protein of the present invention is strongly expressed in liver, ovary and testis. There are many different types of collagen in the body, which together with other extracellular matrix components such as elastin, gelatin, proteoglycans and fibronectin, make up a large proportion of the extracellular tissue of the body. Matrix metalloproteinases (MMPs) are enzymes involved in the degradation and denaturation of extracellular matrix components. Collagenase is, for example, MMP that degrades or denatures collagen. It is known that there are many different collagenases. These include interstitial collagenases, type IV-specific collagenases and collagenolytic proteinases. Collagenase is generally specific in its complete triple-helix structure to collagen, which is extremely resistant to other enzymes. Other MMPs are involved in the degradation and denaturation of different extracellular matrix components such as elastin, gelatin and proteoglycans. Certain MMPs are capable of degrading or degrading several different types of collagen and other extracellular matrix components. For example, stromelysin degrades type IV collagen present in the basement membrane and also affects other extracellular matrix components such as elastin, fibronectin and cartilage proteoglycans. Due to their ability to degrade various components of connective tissue, MMP metalloproteinases such as collagenase, stromelysin and gelatinase are potential targets for controlling numerous pathological processes.

The presence of tissue inhibitors of MMPs was observed in monolayer cultures of various explants and mammalian connective tissue cells (Vater et al., 1979 and Stricklin and Wegus 1983).
. In addition to collagenase inhibitors, other inhibitors of MMPs have also been discovered, eg gelatinases and proteoglycanases. MMP inhibitors generally cannot bind the inactive (enzyme precursor) form of their respective enzymes, but readily complex with the active form (Murphy et al., 1981). Tissue MMP inhibitors include, for example, dermal fibroblasts, human lung, gingiva, tendon and corneal fibroblasts,
Found in human osteoblasts, uterine smooth muscle cells, alveolar macrophages, amniotic fluid, plasma, serum and alpha granules of human platelets (Stricklin and Wegus 1983; We.
lgus et al. 1985; Welgus and Stricklin 1983; Bar-Sharvit et al., 1985; Wooley et al., 1976 and Cooper et al., 1985). The protein of the present invention is a secreted TIMP-1 protein that forms a complex with metalloproteinase in close contact with it and irreversibly inactivates it. TIMP-1 has been identified as a secretory product of platelets and alveolar macrophages. Thus, embodiments of the present invention relate to the use of the proteins of the present invention or fragments thereof to inhibit the action of MMPs by directly inhibiting the enzymatic activity like conventional inhibitors. The inhibitory activity of MMP inhibitors can be assessed by any method suitable for measuring the inhibitory activity of a compound with respect to an enzyme. Such methods are described in standard biochemistry textbooks. In one embodiment, the protein of SEQ ID NO: 291 is rheumatoid arthritis, an inflammatory disorder such as osteoarthritis, osteopenia such as osteoporosis, emphysema, periodontitis, gingivitis, corneal epithelial or gastric ulceration, And for the treatment and diagnosis of disorders associated with overexpression of MMPs such as tumor metastasis, invasion and growth, Paget's disease, hyperparathyroidism and the like. MMP inhibitors may also be used to treat neuroinflammatory disorders, including those associated with myelin degradation, such as multiple sclerosis, as well as arthritic conditions and solid tumor growth and psoriasis, proliferative retinopathy, neovascularization. It may be useful in the management of angiogenesis-dependent diseases including glaucoma, eye tumors, angiofibromas and hemangiomas. The present invention relates to atherosclerotic plaque rupture, restenosis, aortic aneurysm (including abdominal aortic aneurysm and cerebral aortic aneurysm), congestive heart failure, left ventricular dilation, myocardial infarction, decubitus ulcer, chronic ulcer and wound, renal disease, Or other autoimmune or inflammatory disease that depends on tissue infiltration by leukocytes, Crohn's disease, acute respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, Alzheimer's disease, organ transplant toxicity, cachexia, allergic reaction, allergic Contact hypersensitivity, epidermolysis bullosa, loosening of artificial joint implants, stroke, cerebral ischemia, head injury, spinal cord injury, neurodegenerative disorders (acute and chronic), Huntington's disease, Parkinson's disease, migraine, depression, peripheral Neuropathy, pain, cerebral amyloid angiopathy, nootropic or cognitive enhancement, amyotrophic lateral sclerosis, ocular angiogenesis, macular degeneration, abnormal wound healing , A method for treating diseases associated with MMP such as burns, diabetes, scleritis, AIDS, sepsis and infectious shock.

In another embodiment, the protein of SEQ ID NO: 291 has potential utility in the treatment or diagnosis of atherosclerosis. The rupture of atherosclerotic plaque
It is the most common event that causes coronary thrombosis. Destabilization and degradation of the extracellular matrix surrounding these plaques by MMPs has been proposed as the cause of plaque cracking. The shoulders and regions of foam cell accumulation in human atherosclerotic plaques show a local increase in gelatinase B, stromelysin 1 and interstitial collagenase expression. In situ zymography of this tissue revealed increased gelatinolytic and caseinolytic activity (Galla et al., J. Clin. Invest.,
1994; 94: 2494-2503). In addition, high levels of stromelysin RNA messages were found to be localized to individual cells in atherosclerotic plaques removed during surgery from heart transplant patients (Henney, et al., Proc Nat'l. Acad. . Sci., 19
91; 88: 8154-8158). In another embodiment, the proteins of the invention have utility in the treatment or detection of degenerative aortic disease associated with thinning of the central aortic wall. Increased levels of MMP proteolytic activity have been identified in patients with aortic aneurysms and aortic stenosis (Vine N. and Powell JT, Clin Sci., 1991; 81: 233-239).
). In other embodiments, the proteins of the invention can be used as a therapeutic or diagnostic tool for heart failure and its associated ventricular dilation. Heart failure develops by a variety of etiologies, a common feature of which is diastole, which is recognized as an independent risk factor for mortality (Lee et al., Am. J. Cardiol., 1993; 72: 672. -676). This remodeling of the failing heart appears to be associated with extracellular matrix breakdown. MMPs are increased in patients with both idiopathic and ischemic heart failure (Reddy et al., Clin
Res., 1993; 41: 660A; Tyagi SC et al., Clin Res., 1993; 41: 681A).
In animal models of heart failure, induction of gelatinase is important in diastole (Arms
trong et al., Can. J. Cardiol., 1994; 10: 214-220), and diastole precedes a marked lack of cardiac function (Sabbah et al., Am. J. Physiol., 1992; 263: H266-). H27
0). In other embodiments, the proteins of the invention are useful in the treatment or detection of neointimal hyperplasia that often occurs after coronary reconstructive surgery and results in restenosis. Migration of vascular smooth muscle cells (VSMCs) from the media to the neointima is a key event in the highly predictable occurrence and progression of many vascular diseases and the consequences of mechanical injury to blood vessels ( Bendeck MP et al., Circulation Research, 1994; 75: 539-545). Northern blotting and zymography analysis showed that gelatinase A was the major MMP expressed and excreted by these cells. In addition, antisera capable of selectively neutralizing gelatinase A activity also inhibited VSMC migration across the basement membrane barrier. (Pauly RR et al., Circulation Research, 1994; 75: 41-54).
.

In another embodiment, the proteins of the invention are used to identify normal renal function that is dependent on differentiation as well as maintenance of tissues constructed from highly specialized renal cells. The cells are dynamically in equilibrium with surrounding extracellular matrix (ECM) components (Davies M. et al., Kidney Int., 1992; 41: 671-678). Efficient glomerular filtration requires a semipermeable glomerular basement membrane (GBM) composed of collagen, fibronectin, enactin, laminin and proteoglycans. Structural equilibrium is achieved by equilibrating the sustained precipitation of ECM proteins by degradation by specific MMPs. These proteins are initially secreted as zymogens and later activated in the extracellular space. These proteinases
A naturally occurring inhibitor such as TIMP is balanced by offsetting its regulation of activity. Deficiencies or defects in filtration barrier components have important consequences for long-term renal function. For example, in Alport's hereditary nephritis, which is associated with mutations in the gene encoding the ECM protein, defects in collagen assembly result in progressive renal failure associated with cleavage of GBM, ultimately in the glomerulus and interstitium. This produces qualitative fibrosis.
In contrast, in inflammatory kidney diseases such as glomerulonephritis, cell proliferation of glomerular components often precedes apparent ultrastructural changes in the ECM matrix. Cytokines and growth factors involved in proliferative glomerulonephritis such as interleukin 1, tumor necrosis factor, and transforming growth factor beta can upregulate metalloproteinase expression in renal mesangial cells (Martin J. et al., J. et al. Immunol
., 1986; 137: 525-529; Marti HP et al., Biochem J., 1993; 291: 441-446.
Marti HP et al., Am. J. Pathol., 1994; 144: 82-94). These metalloproteinases are characterized by abnormal tissue remodeling and renal disease, such as IgA nephropathy, which progresses the process of progressive glomerular fibrosis and lacks GBM function leading to the final stages of renal disease. It is thought to be closely involved in specific cell proliferation. Metalloproteinase expression has already been well characterized in experimental immune complex-mediated glomerulonephritis such as the anti-Thy 1.1 rat model (Bagchus WM et al., Lab. Invest., 1
986; 55: 680-687; Lovett DH et al., Am. J. Pathol., 1992; 141: 85-98).
.

In another embodiment, the proteins of the invention can be used as a therapeutic or diagnostic tool for gingiva. Collagenase and stromelysin activity have been demonstrated in fibroblasts isolated from inflamed gingiva (Uitto VJ et al., J. Periodontal R
es., 1981; 16: 417-424), and enzyme levels correlated with the severity of gum disease (Overall CM et al., J. Periodontal Res., 1987; 22: 81-88). In other embodiments, the proteins of the invention are useful in treating or detecting ulcers. Proteolytic degradation of extracellular matrix has been observed in corneal ulceration after alkaline burns (Brown SI et al., Arch Opthalmol., 1969; 81: 370-373).
). Thiol-containing peptides inhibit collagenase isolated from alkaline burned rabbit corneas (Burns FR et al., Invest. Opththamol., 1989; 30: 1569-157.
Five). Stromelysin, a member of the MMP family, is produced by basal keratinocytes in various chronic ulcers (Saarialho-Kere UK et al., J
Clin. Invest., 1994; 94: 79-88). Stromelysin 1 mRNA and protein were detected in adjacent basal keratinocytes distant from the wound margin, probably representing sites of epidermal proliferation. Stromelysin 1 may therefore prevent epidermal healing. In other embodiments, the proteins of the invention can be used as a therapeutic or diagnostic tool for tumor angiogenesis. Inhibitors of MMPs showed activity in tumor angiogenesis models (Taraboletti G. et al., Journal of the National Cancer Institute.
, 1995; 87: 293; and Benelli R. et al., Oncology Research, 1994; 6:25.
1-257). Davies et al. (Cancer Res., 1993; 53: 2087-2091) reported that the peptide reduces tumor burden and prolongs survival of mice bearing human ovarian cancer xenografts. The peptide, a conserved MMP propeptide sequence, is a weak inhibitor of gelatinase A and inhibits the invasion of human tumor cells through the reconstructed basement membrane layer (Melchiori A. et al. Cancer Res., 1992; 52: 2353-2356), as well as a natural tissue inhibitor of metalloproteinase 2 (TIMP-2), also showed blockade of tumor cell invasion in an in vitro model (DeClerck YA et al. Cancer Res., 1992).
52: 701-708). Human cancer studies show that gelatinase A is present on the surface of infiltrating tumor cells.
(Strongin AY et al., J. Biol. Chem., 1993; 268: 14033-14039).
), And retained there through interaction with a receptor-like molecule (Monsky W. L. et al. Cancer Res., 1993; 53: 3159-3164).

In another embodiment, the proteins of the invention can be used in the treatment and diagnosis of rheumatoid arthritis. Collagenase has been implicated in several diseases, including rheumatoid arthritis (Mullins, DE et al. 1983), and the use of MMP inhibitors for the treatment of this condition has been proposed. Several investigators have demonstrated a consistent elevation of stromelysin and collagenase in synovial fluid from rheumatoid and osteoarthritic patients when compared to controls (Walakovits LA et al., Arthritis Rheum., 1992; 35). : 35-42; Za
farullah M. et al., J. Rheumatol., 1993; 20: 693-697). TIMP-1 and TIMP-2
Prevented the formation of collagen fragments, but not proteoglycans, from the degradation of both bovine nose and porcine articular cartilage models for arthritis, whereas the synthetic peptide hydroxamate was able to prevent the formation of either fragment ( Andr
ews HJ et al., Biochem. Biophys. Res. Commun., 1994; 201: 94-101). In another embodiment, the proteins of the invention are used in the treatment or diagnosis of inflammation. Gijbels et al. (J. Clin. Invest., 1994; 94: 2177-2182) recently described a peptide that suppressed the development of experimental allergic encephalomyelitis (EAE) and reversed its clinical expression in a dose-dependent manner. , Which suggested the use of MMP inhibitors in the treatment of autoimmune inflammatory diseases such as multiple sclerosis. A recent study by Madri elucidated the role of gelatinase A in T cell extravasation from the inflamed bloodstream (
Ramanic AM and Madri JA, J. Cell Biology, 1994; 125: 1165-1178).
. This transmigration through the endothelial cell layer is coordinated by the induction of gelatinase A,
It is mediated by binding to vascular cell adhesion molecule (VCAM-1). Once the barrier is compromised, the CNS develops edema and inflammation. Leukocyte migration across the blood-brain barrier is known to be associated with the inflammatory response in EAE. Inhibition of the metalloproteinase, gelatinase A, blocks the degradation of extracellular matrix by activated T cells required for CNS penetration. These studies show that inhibitors of stromelysin 1 and / or gelatinase A cause inflammation due to lymphocyte infiltration, inappropriate migration of metastatic or activated cells, or loss of structural integrity required for organ function. Provide a basis for the conviction to treat diseases associated with the destruction of extracellular matrix caused by. The present invention provides the use of MMP inhibitors in the manufacture of a medicament for the treatment or prevention of scarring. Collagen is a major component of scars and other contractile tissue, which is the most important structural component to consider. Contraction of tissues containing extracellular matrix components, particularly collagen-containing tissues, can occur in the context of many different pathological conditions and surgical or cosmetic procedures. Contractures, such as scarring, can cause physical problems that require medical attention or can cause problems from a purely cosmetic perspective.

During experiments in an in vitro model of scar contraction, collagen appears to be infiltrated by fibroblasts and permanently remodeled, and such infiltration or remodeling is likely suppressed by collagenase inhibitors. . Remodeling is generally collagen contraction, which can be suppressed by inhibition of collagenase. Moreover, inhibition of other MMPs also suppresses contraction. The present invention also relates to the use of MMP inhibitors in the treatment or prophylaxis of natural or artificial tissues containing extracellular matrix components that suppress, ie limit, prevent or prevent the contraction of tissues, in particular those caused by pathological conditions or surgical or cosmetic treatments. To supply. Cosmetic treatments, such as chemical or physical abrasion of the skin, used as an anti-aging treatment cause trauma to the skin. The use of MMP inhibitors during the healing process that occurs after initial wear is a cosmetic use of MMP inhibitors according to the invention. The invention also provides for the invasion by cells, in particular fibroblasts, into tissues containing extracellular matrix and / or the migration by cells, especially fibroblasts, in or through tissues containing extracellular matrix. To suppress, ie limit, obstruct, or prevent
Supply applications for MMP inhibitors. In another embodiment, the protein is used to prevent or reduce contraction of scar tissue due to eye surgery. Glaucoma surgery to create new drainage channels often fails due to tissue scarring and contraction. Methods of preventing the contraction of scar tissue formed in the eye, such as the application of appropriate drugs, are therefore valuable.
Such agents can also be used to control corneal trauma or corneal surgery, eg, contraction of scar tissue formed after a laser or surgical procedure for myopia, or refractive errors in which tissue contraction results in inaccurate results. It also results, for example, in some diabetics ultimately resulting in blindness, and when scar tissue formed after dissection, called proliferative vitreoretinopathy, forms in / on the vitreous humor or retina. Useful for. Other uses include scar tissue formed in the orbit or on the eye and eyelid muscles after strabismus, orbital or eyelid surgery, thyroid eye disease, and conjunctival scarring, glaucoma surgery or scar diseases, inflammatory diseases such as Includes pemphigoid or cases that occur following an infection, such as trachoma. A further ocular problem associated with contraction of collagen-containing tissue in which the methods and drugs of the present invention can be used is lens opacification and contracture after cataract extraction.

In a preferred embodiment, the proteins of the invention can be used to treat burns. The contraction of collagen-containing tissue, which may include other extracellular matrix components, often results in
It occurs in the healing of burns. Burns can be chemical, thermal, or radiant burns,
It can be the eye, the surface of the skin or the skin and the underlying tissue. For example, burns due to radiation treatment can be internal tissue. A further aspect of the invention is the inhibition of skin graft contraction. Skin grafts are applied for a variety of reasons and often experience contractions after application. With the healing of burn tissue, contraction becomes both a physical and cosmetic problem. This is a particularly serious problem when a large number of skin grafts are required, such as in severe burn cases. A relevant area in which the drugs and methods according to the invention find excellent use is in the production of artificial skin. To create true artificial skin it is necessary to have an epidermis consisting of epithelial cells (keratinocytes) and a dermis consisting of collagen in which fibroblasts are present. Having both these types of cells is important because they signal and stimulate each other with growth factors. The main problem to date is that collagen components in artificial skin are often in the primary region occupied by fibroblasts.
It was to shrink less than 1/10. MMP inhibitors, such as collagenase inhibitors, can be used to suppress contraction to the extent that artificial skin can be maintained at a practical size. [Protein of SEQ ID NO: 276 (157-15-4-0-B11-CS)] The protein of SEQ ID NO: 276 encoded by the cDNA of SEQ ID NO: 35 is a mutant testis-specific isoform of human calpastatin. . The protein of SEQ ID NO: 276 has two potential transmembrane segments (positions 5-25 and 109-129) predicted by the software TopPred II (Claros and von Heijne, CABIOS applic N
otes, 10: 685-686 (1994)), and signal peptide (8 position LAVILTLLGLAI)
L / AI) is included. Like the human calpastatin protein (Genseq accession number W19395), the protein of SEQ ID NO: 276 is overexpressed in testis. Calpastatin is a physiological inhibitor of calpain. Calpains, a group of ubiquitous Ca2 + -activated cytosolic proteases, are proteolytic by cytoskeletal remodeling events, cell adhesion, shape changes, and site-specific regulation of cytoskeletal proteins that associate with membranes and actin. It is thought to be involved in mobility (Beckerle et al., Cell 51: 569-577, 1987; Yao et al., Am. J. Physiol. 2).
65 (pt. 1): C36-46, 1993; and Shuster et al., J. Cell Biol 128: 837-848.
, 1995). Calpain is also involved in the pathophysiology of brain and myocardial ischemia, platelet activation, NF-kB activation, Alzheimer's disease, muscular dystrophy, cataract progression and rheumatoid arthritis. Cell adhesion, cytoskeletal remodeling events and cell mobility are of considerable interest for inhibitors of calpain as they are associated with a number of pathological conditions (Wang et al., Trends in Pharm. Sci. 15: 412-419, 1994. ; Mehdi, Trend
S. Biochem. Sci. 16: 150-153, 1991). In addition, the calpain / calpastatin system is involved in membrane fusion events in several cell types and, in addition, calpain is detected in human sperm and testis by Western blotting with specific antisera, thus making tCAST fertilizable. It can modulate calpain in a calcium-mediated acrosome reaction that is required (Li S et al., Biol Reprod, 63 (1): 172-
8, 2000).

Calpastatin consists of a unique N-terminal domain (L domain) and four repetitive protease inhibitor domains (domains 1-4) (Lee WJ et al., J Bio.
Chem, 267 (12): 8437-42, 1992). Isolated cDNAs from various mammalian species have significant differences in the region encoding the N-terminal sequence and can be classified into four types. Alternative splicing is probably responsible for molecular diversity, with multiple isoforms involved in specific physiological roles (Lee WJ et al., J Biol Chem, 267 (12): 8437-42.
, 1992). A short isoform, type IV (or human tCAST), is specifically expressed in testis (Takano J et al., J Biochem Tokyo; 128 (1): 83-92, 200).
0). Human tCAST consists of an N-terminal T domain that is 40 amino acids in length and part of domain II derived from somatic isoforms, all of domains III and IV. The protein of SEQ ID NO: 276 shows extensive homology to the N-terminal region of the testis basic-specific protein (U60665 and human calpastatin protein (W19395). The homology region is the T domain of human calpastatin protein (W19395). And domain II. The T domain targets cytosolic localization and membrane association of tCAST, while domain I of somatic calpastatin protein (sCAST) exhibits a nuclear localization function (Li S Et al, Bio
l Reprod, 63 (1): 172-8, 2000). The protein of SEQ ID NO: 276 is a member of the calpastatin family,
It is thought to be involved in cytoskeletal remodeling events, cell adhesion, shape changes, and proteolytic mobilities by site-specific regulation of cytoskeletal proteins that associate with membranes and actin. A preferred polypeptide of the invention is a polypeptide comprising amino acids of sequence 276 from positions 1-119. Other preferred polypeptides of the present invention are fragments of sequence 276 that have any of the biological activities described herein. One embodiment of the invention relates to methods of using the proteins of the invention, or portions thereof, in assays for detecting the presence of calpain in body fluids, tissue samples, or biological samples such as mammalian cell cultures. Since calpastatin can bind calpain (Murachi, Biochemistry Int., 18 (2) 263-294, 1989), the proteins of the present invention can be used to test for the presence of calpain using techniques known to those of skill in the art. Can be used in assays and diagnostic kits. Preferably, a defined amount of the protein of the present invention or a portion thereof is added to the sample under conditions capable of forming a complex between the protein of the present invention or a portion thereof, and the complex and / or The presence of free protein of the invention or a portion thereof is assayed and compared to a control.
Calpastatin has been shown to be useful as a marker of intracellular calpain activation and can be used to monitor the involvement of calpain in pathological conditions (De Tulli
o, FEBS letter, 475 (1): 17-21, 2000). Calpain is a pathophysiology of disorders such as ischemia and Alzheimer's disease (Wronski et al., J. Neural transm.,
107 (2): 145-157, 2000), apoptosis in rat nerve cells with spinal cord injury (SCI) (Ray, Brain res., 867 (1-2): 80-9, 2000), cell fusibility (Kos
ower et al., Methods Mol Biol., 144: 181-94, 2000) and others involved in cytoskeletal proteolysis associated with physiopathology. Assays that detect increased levels of calpain in cells therefore allow for the diagnosis of the diseases or conditions described herein. Furthermore, recent studies have shown that, in addition to its proteolytic activity on cytoskeletal proteins and other cellular regulatory proteins, the calpain-calpastatin system can also influence the expression level of genes encoding structural or regulatory proteins. (Chen et al., Am. J. Physiol. Cell Physiol, 279: C709-C7.
16, 2000). Therefore, the ability to detect levels of calpastatin and calpain would probably be useful in diagnosing a much larger number of diseases and conditions.

In other embodiments, the polynucleotides or polypeptides of the invention include gametes using any of the techniques known to those of skill in the art, including those involved in the use of specific antibodies and nucleic acid probes. Or it can be used to detect specific structures in gametes. Various studies have shown that calpastatin is found in sperm acrosomes (Li et al., Bio.
63: 172-178, 2000), and more precisely, it was shown to exist between the plasma membrane and the outer acrosomal membrane of cynomolgus monkey sperm (Yudin AI, J Androl, 21 (5): 721-9). ,
2000). The ability to see sperm in general, or sperm acrosome in particular, has obvious utility in some applications, including analysis of infertility in patients, as described below. Another embodiment of the invention relates to a method of inhibiting calpain in a cell. Various studies have shown that it is possible to suppress calpain in a cell-free protease activity assay in a dose-dependent manner, in which the calpain inhibitor cerebrolysin, a rat model of acute cerebral ischemia, Ability to protect microtubules associated with protein 2 (MAP2) (Wronski et al., J. Neural Trans
m Suppl., 59: 263-272, 2000), and E-64-D, a cell permeability and selective inhibitor of calpain, can diminish calpain activity associated with apoptosis in rat SCI (Ray et al. Brain. Res., 867 (1-2) 80-9, 2000). Similarly, the protein of SEQ ID NO: 276 can be used to suppress calpain in vitro or in vivo. Calpain affects brain and myocardial ischemia,
Any of these diseases or conditions are involved because they are involved in several pathological processes, diseases and conditions such as platelet activation, NF-kB activation, Alzheimer's disease, muscular dystrophy, cataract progression and pathophysiology of rheumatoid arthritis. It can be treated or prevented by increasing or decreasing the activity or expression of the protein in mammalian cells affected by the disease or condition. Such an increase involves introducing into a cell a polynucleotide encoding a protein of the invention operably linked to a promoter, and administering to the cell a compound that increases the activity or expression of the protein of the invention. Can be effected in a number of ways, including but not limited to: In addition, expression or activation of the proteins of the invention can be accomplished by a number of methods, including using antisense oligonucleotides, antibodies, dominant negative forms of proteins, and using heterologous compounds that reduce the expression or activation of the invention. It can be suppressed by means. Such compounds can be readily identified by, for example, screening candidate compounds, detecting protein expression or activity levels using standard assays. In another preferred embodiment, the protein of the invention is used for the treatment or diagnosis of infertility,
Alternatively, it can be used to modify and / or characterize fertility, including contraception. Since the calpain / calpastatin system participates in the acrosome reaction, which is a necessary step in fertilization, over- or under-expression or activation of the protein disrupts this reaction, thereby inhibiting fertility. Therefore, the cause of infertility in a large number of patients can probably be detected by detecting the expression level of the protein, and an abnormal level of activity or expression of the protein may indicate that the cause of infertility is a calpain-dependent acrosome. Indicates a reaction. Such a diagnosis also points to methods of treating infertility, for example by increasing or decreasing the expression or activation of the protein in sperm. Alternatively, contraception includes increasing the protein level using, for example, a polynucleotide encoding the protein, the protein itself, or an activator of protein expression or activity, or a dominant antisense oligonucleotide, antibody, or protein. By lowering protein levels using inhibitors such as the negative form, as well as heterologous compounds that suppress protein expression or activity, the expression or activation of the protein can be artificially disrupted.

<Protein of SEQ ID NO: 295 (Internal Designation Number 181-20-3-0-B5-CS)> The protein of SEQ ID NO: 295 encoded by the cDNA of SEQ ID NO: 54 is a rat, bovine, and human uromodulin precursor. It shows homology to the substance Tam-Horsfall urinary glycoprotein and the precursor of the major GP2 glycoprotein of thuman pancreatic secretory granulosa. SEQ ID NO: 295 exhibits homology in the 5'region with both GP2 and uromodulin (40% identical, and 60% similar). Like GP2 and uromodulin, homologous segments contain an EGF-like calcium binding domain, some potential disulfide bonds, and some potential N-linked glycosylation sites. Calcium-binding EGF-like domains contain a calcium-binding site at the N-terminus and have been found in proteins that require calcium for their biological activity. Calcium binding EG
Non-limiting examples of proteins containing F-like domains include (1) coagulation factors X, VII, IX; (2) LDL
A receptor; (3) thrombomodulin; and (4) fibrillin 1. Downin
g et al. <Cell 85: 597-605 (1996)> described a disease-causing mutation that destabilizes a covalent pair of the Ca2 + -binding EGF-like domain of fibrillin-1 (associated with Marfan syndrome). These domains form a strong rod sequence that is stabilized by intradomain calcium binding and hydrophobic interactions. Uromodulin
(URO) is a 90-100 kDa glycoprotein synthesized by the ascending loop of the bladder and the epithelial cells of the curved tubule. Except for glycosylation, URO is the most abundant protein in normal human urine, the Tam-Horsfall protein (THP
) Is the same. The relative abundance and the specific nephron position of URO suggest that it may have important physiological functions in the urinary system. URO is also an immunosuppressive glycoprotein and has been found to inhibit antigen-induced human T cell proliferation. More recent studies have shown that URO triggers the inflammatory response of neutrophils and stimulates human mononuclear cells to proliferate and release cytokines and gelatinases. Uromodulin is associated with recombinant interleukins 1 and 2, as well as tumor necrosis factor (T
It has been shown to be involved in the regulation of circulating activity of cytokines due to its high affinity binding to (NF). URO is a T cell as monitored by lysis of target tumor cells.
It does not inhibit the cytotoxic activity of NFα, but interacts with recombinant TNFα via the carbohydrate chain. This interaction is essential in eliminating and / or promoting the in vivo toxicity of TNFα and other lymphokines. Endotoxin shock and sepsis are due to the cytokines IL-1 and TNFα. URO is IL-1 and
URO may be effective as a therapeutic agent for these conditions because it appears to exhibit inhibitory activity against TNFα. Uromodulin may also be involved as an inhibitor of certain bacterial infections in the bladder and urethra. URO
Has the ability to bind to type 1 fimbriae of E. coli and prevent attachment to epithelial surfaces.

SEQ ID NO: 295 also has homology to glycoprotein GP2. GP2 is a zymogen complex protein of the pancreatic granulosa. GP2 is anchored to the lipid bilayer via a glycosylphosphatidylinositol (GPI) bond and released by the calcium-activating enzyme into the zymogen granule contents. Through the exocytosis process, GP2 is released into the pancreatic duct. The protein is also soluble within the zymogen, which is stored in the granules, and is secreted by the pancreas and detected in pancreatic secretions. GP2 appears to be involved in the development of pancreatitis, an inflammation of the pancreas that involves autolysis of pancreatic tissue by its own enzymes. After cloning and sequencing GP2, Gen
A search of the bank database showed one homologous protein, uromodulin. Studies have shown that GP2 and URO not only share structural homology, but are functionally identical, so that both may form vascular precipitates under pathological conditions. Aggregation of these precipitates in the pancreas causes obstruction of the pancreatic duct and may play a critical role in the development of pancreatitis. Similarly, URO aggregation in the kidney can cause renal tubular obstruction, resulting in renal disease. The invention provides a protein of SEQ ID NO: 295 and a polynucleotide sequence encoding SEQ ID NO: 295. The invention also includes bioactive fragments of the protein encoded by SEQ ID NO: 295, and polynucleotide sequences encoding these bioactive fragments. A "bioactive fragment" is a SEQ ID NO: that has at least one of the biological functions of a full-length protein (eg, the ability to chelate calcium, bind to E. coli pili, or cause immune regulation in an individual). It is defined as 295 peptides or polypeptides. In one embodiment, the polypeptide of SEQ ID NO: 295 is a human cDN that is clone 181-20-3-0-B5-CS.
Compatible with the polypeptide encoded by A. The invention also provides a variant of SEQ ID NO: 295. These variants have at least about 80%, more preferably at least about 90% of the amino acids of SEQ ID NO: 295.
%, And most preferably at least about 95% amino acid sequence identity. Variants according to the invention also have at least one functional or structural feature of SEQ ID NO: 295, such as the biological function or EGF-like calcium binding domain described above. The present invention also provides bioactive fragments of variant proteins. Unless otherwise stated, the methods disclosed herein can be performed using SEQ ID NO: 295 or variants thereof. Similarly, the methods of the invention can be practiced with the biological fragment of SEQ ID NO: 295, or a variant of said biologically active fragment.

Due to the redundancy of the genetic code, a variety of different DNA sequences can encode SEQ ID NO: 295. It is within the skill of one in the art to create these alternative DNA sequences that encode proteins having identical or substantially identical amino acid sequences. These mutant DNA sequences are therefore within the scope of the invention. As used herein, a "substantially identical" sequence is an amino acid substitution that does not substantially affect biological activity,
By a sequence with deletions, additions or insertions is meant. A "recombinant nucleotide variant" is an alternative polynucleotide that encodes a particular protein. They can be synthesized, for example, by taking advantage of the "redundancy" of the genetic code. Diverse codon substitutions, such as silent changes that produce specific restriction sites or codon usage-specific mutations, optimize cloning into plasmid or viral vectors, or expression in specific prokaryotic or eukaryotic host systems, respectively Can be introduced to SEQ ID NO: 295 and variants thereof can be used to raise antibodies by methods known in the art. Antibodies can also be synthesized by known methods against fragments of SEQ ID NO: 295 as well as variants of SEQ ID NO: 295. The invention also provides an antibody that specifically binds to a bioactive fragment of SEQ ID NO: 295 or a variant bioactive fragment of SEQ ID NO: 295. The present invention also provides immunoassays used to screen, monitor, or diagnose conditions or disorders associated with liver dysfunction and / or injury. These include, but are not limited to, hepatitis, cirrhosis, fibrosis, periductal inflammation, portal triaditus, chronic periportal inflammation, systemic lupus erythematosus, Hodgkin's disease, granulomas and cell dysplasia The condition or disorder can also be diagnosed.
For some disorders listed above, significantly higher levels of these genes compared to standard gene expression, eg, expression levels in healthy tissue or body fluids from one or more individuals not suffering from the disorder, or Low expression levels have been obtained from individuals with such disorders in particular liver tissues or cell types (eg, cancerous),
Alternatively, it can be routinely detected in body fluids such as serum, plasma, and blood.
These types of assays allow non-invasive methods of screening, diagnosing, or monitoring liver cancer in human subjects. Similarly, antibodies or small molecules directed against the polypeptide can be used as immunological probes for the taxonomic identification of diseased tissues or cells.

[0186]   In addition, the nucleic acid and amino acid sequences of SEQ ID NOs: 54 and 295 are
Or the polypeptide and its biological activity for repair of cell injury after liver transplantation
Can be used to supply fragments.   In addition, the polypeptide, or biologically active fragment thereof, can be targeted to epithelial cells.
Can be used as a modulator of fungal binding or as a modulator of bacterial infection
. In this aspect of the invention, the bacterial cells are sufficient to prevent bacterial binding to epithelial cells.
Contact with a composition comprising a sufficient amount of the polypeptide, or bioactive fragment thereof
To do. In one embodiment, the bacterium is an E. coli type bacterial cell. In other embodiments
, The bacterial cell is E. coli. SEQ ID NO: 295 or a biologically active fragment thereof
The composition is administered in any manner needed to provide a therapeutic effect.
(Eg, oral, intravenous, intrathecal, intraarterial, etc.).   The invention also relates to substances and agents for the treatment of endotoxic shock and / or sepsis.
And supply method. In this embodiment, the subject has a therapeutically effective amount of SEQ ID NO: 295 or
Is treated with a composition comprising the bioactive fragment.   The present invention also provides calcium ions (Ca²⁺) In vivo or in vitro killers
Supply materials and methods for conversion. In this aspect of the invention, SEQ ID NO: 295
Or a biologically active fragment thereof, in a solution, environmental sample, or biological sample,
By adding a polypeptide, or a bioactive fragment thereof, free Ca ²⁺  Can be used to combine Alternatively, SEQ ID NO: 295, or biological activity thereof
The composition containing the fragment is transferred from the solution to the solution, environmental sample, or biological sample.
Sufficient amount can be added to bind and remove detached Ca 2+.   In another aspect of the invention, SEQ ID NO: 295, or a biologically active fragment thereof, is
It can be used to modulate the immune system of a mammal. In this way,
Of SEQ ID NO: 295, or a bioactive fragment thereof, in an acceptable carrier.
An immunomodulatory amount can be administered to the mammal. The method of assessing the stimulatory state of the mammalian immune system is
It can be performed according to a method known in the art.   <Proteins of SEQ ID NOs: 244 and 251 (internal designation number 105-016-3-0-G10-CS
And 105-074-3-0-H10-CS)>   A 274 amino acid long SEQ ID NO: 244 tamper encoded by the cDNA of SEQ ID NO: 3.
The cytoplasm was found in the prostate gland and was strongly expressed in the salivary glands.
Between the doria carrier protein PET8 (SWISSPROT accession number P38921) and eukaryotes
Conserved similar protein (D. melanogaster)
And C. elegans received SPTREMBLNEW, SPTREMBL and SWISSPROT respectively.
Accession numbers Q9VBN7 and Q18934, and S. pombe: SWISSPROT Accession number Q1044
It shows strong sequence similarity with 2). Mitochondrial carrier / transport protein super
-All members of the family have sequences highly similar to P-X-D / E-X-X-K / R-X-R
Presents a motif, which also has three positions (26-33, 1 in the protein of the invention.
Have been found at positions 08-115, and 199-206) (Belenkiy et al., Biochim. Bi
Ophys. Acta, 1467: 207-218 (2000)). These mitochondrial carrier proteins
The quality signature is associated with a transmembrane segment (Belenkiy et al., Ibid; Kuan et
 Saier, Crit. Rev. Biochem. Mol. Biol., 28: 209-233 (1993)). Actually four
The four transmembrane segment candidates are 4-24, 51-71, 180-200 and 240-260.
Identified in the protein of the invention from the mino acid position. Other hydrophobic areas
Has been found at amino acid positions 86-107 and 139-162. In addition, SEQ ID NO: 2
The 44 proteins have putative signal peptides at the amino acid terminal part (positions 5 to 19).
Indicates the code.

The protein of SEQ ID NO: 251 encoded by the cDNA of SEQ ID NO: 10 is a 72 amino acid long truncated form of the protein of SEQ ID NO: 244. This short product has a 110 bp exon (275-384 positions) present in the cDNA of SEQ ID NO: 3 in the cDNA of SEQ ID NO: 10.
Due to not being present in NA. Nonetheless, the protein encoding 72 amino acids is a putative signal peptide (positions 5-19), a first mitochondrial carrier protein signature (positions 26-33), and two candidate transmembrane segments (4-4).
24 and 51-71 positions). Energy transfer in mitochondria requires the transport of many specific metabolites across this inner membrane of eukaryotic organelles. Different types of matrix carrier proteins involved in energy transfer have been found in the inner membrane. These proteins are all evolutionarily related and are mitochondrial carrier / transport proteins (M
CP / MTP) constitutes the superfamily. Structurally, MCP / MTP proteins are generally homodimeric complex transmembrane polypeptides (subunit molecular weight ~ 30 kD).
It has both N- and C-termini localized to the cytosolic side of the membrane and traverses the inner mitochondrial membrane 6 times. Each 30 kD subunit is composed of three tandem repeats of a domain of approximately 100 residues (-10 kD). This 10 kD domain contains two transmembrane regions and a sequence motif highly similar to PX ₁ -D / EX ₂ -X ₃ -K / RX ₄ -R, where X ₃
Is a hydrophobic residue (Kuan et Saier, Crit. Rev. Biochem. Mol. Biol., 28
: 209-233 (1993)). Five protein families with known functions have been identified in the mitochondrial carrier protein superfamily: (1) One-to-one cytosolic ADP to matrix ATP across the inner mitochondrial membrane under oxidative phosphorylation conditions. ADP, ATP carrier protein (
ACC), ADP / ATP transposase (Fiore et al., Biochimie, 80: 137-150 (1998)). ADP /
The ATP transport system is highly specifically blocked by two families of inhibitors, one of which is atractyloside (ATR) and carboxyatractyloside (CATR), the other of which is boncrecic acid (BA) and isoboncrecine. Acid (isoBA)
Is. It has been confirmed that these inhibitors recognize two different conformations of the carrier protein, CATR and BA conformations, which exhibit different chemical, immunochemical, and enzymatic reactivities. Bakker and coworkers reported that myopathy may be due to defective ADP / ATP transport (Bakker et al., Pediatr. Res. 33:41.
2-417 (1993)). That is, the authors explain that ADP / ATP carrier protein concentrations are four-fold lower in patients with mitochondrial myopathy.

(2) 2-oxoglutarate / malate carrier protein (OGCP), which carries 2-oxoglutarate into the cytosol and malate or dicarboxylic acids into the mitochondrial matrix. This protein plays an important role in several metabolic processes, such as malate / aspartate and oxoglutarate / isocitrate shuttles (Palmieri et al., J. Bioenerg.
Biomembr. 25: 493-501 (1993)). (3) A phosphate carrier protein that transports inorganic phosphate groups from the cytosol into the mitochondrial matrix (Palmieri et al., Ibid.). (4) Mammalian brown fat uncoupling proteins such as UCP-1 (thermogenin)
It is a transmembrane hydrogen ion translocation protein present in brown adipose tissue, a specialized tissue that functions for heat generation and energy balance ((Jezek and Garlid, Int.
J. Biochem. Cell. Biol. 30: 1163-1168 (1998); Klingenberg, J. Bioener
g. Biomembr. 31: 419-430 (1999); Nicolls and Locke, Physiol. Rev. 64: 2-4.
0 (1994); Rothwell and Stock, Nature 281: 31-35 (1979)). Oxidation of substrates by mitochondria involves transport of hydrogen ions from the mitochondrial matrix, creating a transmembrane hydrogen ion gradient. Reentry of hydrogen ions into the matrix, generally via ATP synthase, is coupled to ATP synthesis. But UCP
-1 functions as a transmembrane hydrogen ion transporter, allowing hydrogen ion reentry into the mitochondrial matrix without ATP synthesis. Exposure to low temperature environment induces neural and hormonal stimulation of brown adipose tissue, resulting in UCP
Increases mediated hydrogen ion transport, brown fat metabolic activity and heat production. Studies using transgenic models indicate that brown fat and UCP-1 play important roles in energy expenditure in rodents. Transgenic mice whose brown adipocyte tissue has been cleaved by a toxin linked to the UCP promoter developed obesity and diabetes (Lowell et al., Nature 366: 740-742 (1993)).
Obesity in these transgenic animals occurs in the absence of bulimia, suggesting that brown fat uncoupled mitochondrial respiration is a key component of energy expenditure. In another transgenic mouse model, ectopic expression of UCP-1 in white adipose tissue of genetically obese mice leads to a marked decrease in body weight and fat storage (Kopecky et al., J. Clin. Invest. 96: 2914). -2923 (1995)). These studies show that UCP-1 activity in rodents is associated with energy expenditure and weight loss. Two other UCP proteins have recently been cloned. The first uncoupling protein-like protein (UCPL) or UCP-2 (59% homology) is widely expressed (heart, kidney, lung, placenta and white fat) and further enriched in lymphoid tissues (Fleury et al. , Nature Genetics 15: 269-272 (1997)). Second UC
P-33 (57% homology) is mainly localized in skeletal muscle and brown fat (Boss et al., F
EBS Lett 408: 39-42 (1997)). UCP-3 has been found to be regulated by cold and thyroid hormones (Larkins et al., Biochem. Biophys. Res. Comm.
240: 222-227 (1997)).

Fever protein activity, as found in UCP-1, may be useful in reducing or preventing the development of excess adipose tissue as found in obesity. Obesity is increasing in developed countries. Attempts to reduce food intake or reduce overnutrition are usually less effective because diet-induced weight loss results in increased appetite and reduced energy expenditure. The intensity of physical exertion required to consume sufficient energy to substantially lose fat mass is too great for many obese humans to perform on a sufficiently frequent basis. Therefore, obesity is currently difficult to treat,
It is a chronic, substantially refractory metabolic disorder. In addition, obesity is a severe risk of simultaneous pathology, including increased risk of type 2 diabetes, increased risk of heart disease, hypertension, atherosclerosis, degenerative arthritis, and surgical complications including general anesthesia. With sex. (5) Tricarboxylate transport protein (or citrate transport protein) involved in citrate-H + / malate exchange. This protein is important for hepatocyte bioenergy, as it supplies carbon sources for fatty acid and sterol biosynthesis, and NAD for glycolysis. The protein of SEQ ID NO: 244, or a portion thereof, is a member of the mitochondrial carrier / transport protein superfamily and is therefore capable of crossing the inner membrane of ADP / ATP.
, Malate / aspartate, 2-oxoglutarate / isocitrate,
Citrate-H + / malate exchange, phosphate transport and body weight, energy balance,
It is involved in mitochondrial processes such as thermogenesis that prevents muscle chorea, fever, and defense against reactive oxygen species generation. Preferred polypeptides of the present invention are, on the one hand,
Amino acids of SEQ ID NO: 244 from 26-33, 108-115 and 199-206 amino acid positions and, on the other hand, 4-24, 51-71, 86-107, 139-162, 180-200 and 240-260 amino acid positions. Is a polypeptide containing. Other preferred polypeptides of the present invention are fragments of SEQ ID NO: 244 having any of the biological activities described herein. The protein of SEQ ID NO: 251 is SEQ ID NO: 244.
It is a 72-amino acid truncated form of the protein of Escherichia coli and corresponds to a subunit of the tripartite structure of the mitochondrial carrier / transport protein. Preferred polypeptides are those comprising the amino acids of SEQ ID NO: 251 from positions 4-24, 26-33 and 51-71.

The activity of the protein of the present invention can be evaluated using cultured cells. For example, a nucleic acid encoding the protein of SEQ ID NO: 244 can be cloned into a eukaryotic vector and transfected into a cell population. Transfected mammalian cells are then loaded with, for example, ADP, dicarboxylic acids, inorganic phosphate groups, or H ^{+ into} the mitochondrial matrix, as well as ATP, 2-oxoglutarate, tricarboxylate-H ⁺ sites. It is tested for its carrier activity, such as export into a sol. These transfected cell lines were atractyloside (ATR), carboxyatractyloside (CATR), described above in connection with the ADP / ATP mitochondrial carrier,
Allows the development of in vitro assays for the identification of modulators of carrier activity such as boncrecic acid (BA) and isoboncrecic acid (isoBA). Such modulators are useful in treating diseases or conditions associated with the proteins of the invention. Other embodiments of the invention include, for example, neuroleptic malignant syndrome (NMS) (Kubo et al.,
Forensic Sci. Int. 115: 155-158 (2001)), Rett Syndrome (Armstrong, Brai
n Dev 14 Suppl: S89-98 (1992)), Alpers disease (Chow and Thorburn, Hum. Re
prod. 15 Suppl 2: 68-78 (2000)) or mitochondrial encephalomyopathy (Handran
Et al., Neurobiol. Dis. 3: 287-298 (1997)) in order to visualize alterations in the number, topology, or morphology of this organelle in the context of mitochondria-related human diseases, such as Relates to compositions and methods of using the proteins of the invention or portions thereof to label the inner mitochondrial membrane. For example, the present protein can be easily detected by inserting the protein encoding the cDNA of the present invention into a eukaryotic expression vector having a sequence encoding a tag sequence in frame. Eukaryotic cells expressing the tagged proteins of the invention can also be used for in vitro screening for drugs or genes that allow treatment of mitochondrial-related diseases or conditions. The proteins of the invention can also be used to specifically label salivary glands or prostate cells, for example for histological analysis or identification of the origin of tumor cells. The protein of the present invention also applies radioactive or chemically labeled metabolites (ADP, dicarboxylic acid, inorganic phosphate groups) from the cytosol to the mitochondrial matrix, for example to specifically label mitochondria for the purpose of tracking its modification. It can be used as a carrier / transporter for translocation. For example, radiolabeled or chemically labeled precursors can be added to the in vitro culture of mammalian cells that have been stably transfected to express the proteins of the invention. The labeling of the organelles is then atractyloside (ATR), carboxyatractyloside (CATR), boncrecic acid (BA).
And can be stopped at different times after the start of the experiment by adding a specific inhibitor of the carrier / transport protein, such as isoboncrecic acid (isoBA).
Cells with labeled mitochondria can be used for in vitro screening for drugs or genes that can result in mitochondrial modification.

Another embodiment of the (protein) of the invention or part thereof is to combine a heterologous compound, either a polypeptide or a polynucleotide, with a fragment of the protein of the invention into a heterologous polypeptide or polynucleotide or It relates to a method of delivering to the inner mitochondrial membrane by chemical fusion. Preferred fragments include a putative signal peptide, four transmembrane segments and / or H
Operrion Microbiol 3: 210-4 (200 by errman, Neupert, and Curr.
0); Bhagwat et al., J. Biol. Chem. 274: 24014-22 (1999), Murphy Trends Biot.
echnol 15: 326-30 (1997); Glaser et al., Plant Mol Biol 38: 311-38 (1998);
Other fragments of the proteins of the present invention include targeting signals for mitochondria, including but not limited to matrix targeting signals as defined in Ciminale et al., Oncogene 18: 4505-14 (1999). Such heterologous compounds can be used to modulate mitochondrial activity, such as inducing and / or preventing mitochondria-induced apoptosis or necrosis. For example, these heterologous compounds are associated with immunodeficiency syndromes (including AIDS), type I diabetes, pathogenic infections, cardiovascular and nerve damage, alopecia, aging, Alzheimer's disease, Parkinson's disease, Huntington's chorea, muscle tone. Abnormalities, labels such as inherited visual neuropathy, schizophrenia, muscles such as "mitochondrial encephalopathy, lactic acidosis, and stroke" (MELAS) and "myoclonic tencan-clonic muscle spasm crude red fiber syndrome" (MERRF) It can be used to treat and / or prevent disorders in which apoptosis is detrimental, including but not limited to degenerative disorders. In addition, heterologous polynucleotides can be used to deliver nucleic acids for mitochondrial gene therapy, ie, to replace defective mitochondrial genes and / or to inhibit deleterious expression of mitochondrial genes. The invention further relates to methods and compositions used to modify the proteins of the invention. Post-translational modifications encompassed by the invention include, for example, N-linked or O-linked carbohydrate chains, N-terminal or C-terminal processing, attachment of compound moieties to amino acid backbones, chemical modification of N-linked or O-linked carbohydrate chains, and It includes the addition or deletion of N-terminal methionine residues as a result of prokaryotic host cell expression. These post-translational modifications of the proteins of the present invention may be postulated by methods such as screening of cDNA expression libraries with radiolabeled recombinant proteins, as post-translational modifications are most important in protein-protein interactions. It can be extremely useful in searching for protein partners. Identification of protein partners of mitochondrial carrier proteins allows for studies on their ex vivo and in vivo regulation in normal vs. pathological cases (for example, UCP1 mitochondrial carrier protein and its 14.3.3 physical partners, see Pierrat et al., Eur. J. . Bioch
See em. 267: 2680-2687 (2000)).

Another embodiment of the invention is to establish a transgenic animal model (D. melanogaster, M. musculus) by any of the methods known to those skilled in the art,
Compositions and methods using the polynucleotide sequences encoding the proteins of the invention or portions thereof. The expression of the transgene is controlled by the drug or modified gene (
In vivo modulation with activators or suppressor genes) can develop animal models that mimic human mitochondria-related disorders such as myopathy or obesity. These animal models will therefore allow the identification of potential therapeutic agents for the treatment of disorders. Moreover, recombinant cell lines derived from these transgenic animals can be used ex vivo in a similar manner. In one embodiment, the protein of SEQ ID NO: 251, which corresponds to the 72 amino acid long truncated form of SEQ ID NO: 244, has a dominant negative mutation to suppress the function of the full length form of the protein of SEQ ID NO: 244 in vitro or in vivo. Can be used as a body. Inactivation of mitochondrial carriers in this way allows the development of animal models for human disease. Recently, for example, Lowell and coworkers have shown that in mice, targeted destruction of UCP1 by the diphtheria toxin A chain produces obese animals (Kozak and Koza, ibid, Lowell et al., Ibid). <SEQ ID NO: 285 protein (internal designation number 174-39-2-0-A3-CS)> SEQ ID NO: 285 encoded by the cDNA (clone 174-39-2-0-A3-CS) The protein is overexpressed in cancerous prostate, fetal brain, muscle, and placenta. The protein is homologous to NADH-cytochrome b5 reductase isoform and human electron transfer protein. NADH-cytochrome b5 reductase protein belongs to a family of flavin enzymes that share common structural features and its members (ferrodoxin-NADP + reductase, NA
DPH-cytochrome P450 reductase, NADPH-sulfite reductase, NADPH-cytochrome b5 reductase and NADPH-nitrate reductase) are photosynthetic, nitrogen and sulfur assimilation, fatty acid oxidation, methemoglobin reduction and many insecticides. Involved in the metabolism of drugs and carcinogens (Karplus et al., Science, 251: 60-6 (1991))
. Furthermore, cytochrome b5 reductase appears to play a role in preventing apoptosis after oxidative stress (Villalba et al., Mol Aspects Med 18 Suppll: S7-13.
(1997)).

It is believed that the protein of SEQ ID NO: 285 is a redox enzyme. Therefore,
It is involved in electron transfer and general aerobic metabolism and may be associated with the mitochondrial membrane. Furthermore, the proteins of the invention can use FAD and / or molybdopterin as cofactors. The protein may be involved in photosynthesis, nitrogen and sulfur assimilation, fatty acid oxidation, methemoglobin reduction and metabolism of many insecticides, drugs and carcinogens. A preferred polypeptide of the present invention is a fragment of SEQ ID NO: 285 that has any of the biological activities described herein. The oxidoreductase activity of the proteins of the invention can be assayed using any of the techniques known to those of skill in the art. The ability to bind cofactors is also described, for example, in US Pat.
Assays can be performed using any of the techniques known to those of skill in the art, including the NAD binding assay described in 5,986,172. In other embodiments, the proteins of the invention or portions thereof are used to prevent cells from undergoing apoptosis. In a preferred embodiment, an amount of apoptotic polypeptide that is effective in reducing apoptosis is added to an in vitro culture of mammalian cells. Furthermore, the protein of the present invention or a part thereof is immunodeficiency syndrome (including AIDS), type I diabetes, pathogenic infection, cardiovascular and nerve damage, alopecia, aging, Alzheimer's disease, Parkinson's disease, Huntington chorea. Disease, dystonia, Leber's hereditary visual neuropathy, schizophrenia, “mitochondrial encephalopathy, lactic acidosis, and stroke” (MELAS) and “myoclonic tencan-clonic muscular spasm red fiber syndrome” (MERRF) It can be used to treat and / or prevent disorders in which apoptosis is detrimental, including but not limited to muscle degenerative disorders such as. The invention further includes mitochondrial cell injury, necrosis, aging, neurodegenerative diseases, myopathy, methemoglobinemia, hyperlipidemia, obesity, cardiovascular disorders and cancer, including but not limited to energy metabolism. It relates to methods and compositions using the proteins of the invention or parts thereof for the diagnosis, prophylaxis and / or treatment of some disorders which have been or need to be. For diagnostic purposes, expression of the proteins of the invention may be determined by Northern blot, RT-PCR, as described herein.
, Can be studied using immunoblotting and compared to expression in control individuals. For prophylactic and / or therapeutic purposes, the proteins of the invention can be used to enhance electron transfer as well as increase energy delivery using any of the gene therapy methods described herein.

<Protein of SEQ ID NO: 368 (Internal Designation Number 187-45-0-0-l18-CS)> The protein of SEQ ID NO: 368 encoded by the cDNA of SEQ ID NO: 127 is a polypeptide of 78 amino acids in length. . The sequence of the protein of SEQ ID NO: 368 is a subunit of mammalian oligosaccharyltransferase (OST), which is a protective factor against apoptotic cell death 1 protein, and the sequence of human Dad1 protein and the last 43 residues of Dad1 , The proteins of the present invention are identical except that they are substituted with a series of 8 different amino acids. In addition, the protein of SEQ ID NO: 368 has DA from positions 1-78.
It exhibits the pfam signature of the D family of proteins and two putative transmembrane domains from positions 31-51 and 54-74. Dad1 protein is a 113 amino acid long protein,
Its mRNA consists of three exons <Genbank accession numbers D15057 and Nakashima
, T. et al. (1993) Molecular and Cellular Biology 13: 6367-6374>. The cDNA of SEQ ID NO: 127 consists of the first and third exons of the Dad1 cDNA, but the Dad1 cDN
It lacks the second exon of A. In summary, these data indicate that the protein of SEQ ID NO: 127 is a new isoform of the Dad1 protein due to alternative splicing events. Aspartate-linked glycosylation is a highly conserved protein modification reaction in all eukaryotes. An early stage in the biosynthesis of N-linked glycoproteins, catalyzed by oligosaccharyltransferases (OSTs), is an assembled high-mannose oligosaccharide, a nascent protein in the rough endoplasmic reticulum lumen, from assembled dolichol-linked oligosaccharide donors. Translocation to the aspartate receptor site of <Silberstein, S
.. (1996) FASEB J 10: 849-858>. Protein glycosylation is essential for the structure and function of many proteins and is involved in the control of many diverse biological processes (Paulson, Trends in
Biol Sci., 1989, 14, 272; Sadler, In Biology of Carbohydrates, 2nd
Ed., Ginsburg & Robbins, Ed., John Wiley & Sons New York, 1984, Vol.
2, pg. 87). For example, protein glycosylation is known to be essential for the development, growth and correct functioning of complex organisms, and aberrant glycosylation of proteins is associated with disease or transformed cells. Mammalian oligosaccharyltransferase (OST) consists of four ER membrane proteins, ribophorins I and II (RI and RII), OST48 and DAD1 that form oligomeric complexes.
RI and OST48, and possibly also RII, are type I transmembrane proteins. The luminal domain of OST48 interacts with the domains of RI and RII and the cytoplasmic domain of OST48 has an affinity for the cytoplasmically exposed N-terminal tail of DAD1 <Kelle
her, D. et al. (1997) Proc Natl. Acad. Sci. USA 94: 4994-4999; Fu, J. et al. (1997)
) J. Biol. Chem 272: 29687-29692>.

Dad1 is a small hydrophobic protein and is believed to be an integral membrane protein with an N-terminus located in the cytoplasm and up to 3 transmembrane domains. Like the other subunits of OST, the exact role of Dad1 in N-glycosylation is unknown. However, Dad1 has been shown to be essential for the function and structural integrity of the OST complex <Sanjay, A. et al. (1998) J. Biol. Chem 273: 26094-26099>.
. It is also worth noting that the loss of function of the Dad1 protein was first identified in 1993 as a mammalian cell death suppressor because its loss of function induces apoptosis in hamster BHK21 cells <Nakashima, T. et al., (1993). Molecular and Cellul
ar Biology 13: 6367-6374>. Since then, several reports have confirmed an anti-apoptotic role for Dad1 <Hong, NA. Et al. (2000) Dev Biol 220: 76-84.
Brewster, JL. Et al. (2000) Genesis 26 271-8>; Yoshimi, M. et al. (2000)
Biochem Biophys Res Commun 276: 965-9>. Dad1 is a highly conserved protein that has been sequenced for a variety of organisms, including some vertebrates, nematodes, and some plants. Comparison of these sequences reveals that the amino-terminal region preceding the first transmembrane segment is the minimal conserved region of the protein with respect to both its length and amino acid sequence identity. The most highly conserved sequence of Dad1 contains second and third transmembrane segments and is therefore probably the most important region of Dad1 function <Kelleher, D. et al. (1997) Proc Natl. Acad. . Sci. USA 94: 4994-4999
>. The importance of the C-terminal region for mediating Dad1 function was recently confirmed <Makishima
, T. et al. (2000) J. Biochem (Tockyo) 128: 399-405>. Therefore, it is considered that Dad1 acts as a positive regulator of the oligosaccharyltransferase complex and a negative regulator of apoptosis. Moreover, the C-terminus of the Dad1 protein appears to be important in mediating these functions. As mentioned above, the protein of the invention is a new isoform of the Dad1 protein due to alternative splicing events. As a result of this alternative splicing event, the C-terminus of the protein of the invention is truncated and does not display the third transmembrane domain of Dad1. D
Since the C-terminus of ad1 has been shown to be important in mediating protein function, the proteins of the invention are believed to rather have an antagonistic effect on Dad1 function. It is worth noting that this kind of situation is a common theme among apoptotic genes, where the same genes, which produce different protein products with opposite functions by alternative splicing, are worth noting <For more information, see Reed, JC (1999) Nat. Biotechnol 17:
See 1064-65>.

Thus, the protein of the invention of SEQ ID NO: 368 is involved in the regulation of N-glycosylation of cellular proteins. Preferably, the protein of the present invention will act as a positive regulator of apoptosis and also as a negative regulator of the OST complex. A preferred polypeptide of the invention is a polypeptide comprising the amino acids of SEQ ID NO: 368 from positions 1-78 and 71-78. Other preferred polypeptides of the present invention are fragments of sequence 368, which have any of the biological activities described herein. The activity of a protein of the invention or a portion thereof on protein N-glycosylation can be assayed using any of the assays known to those of skill in the art.
For example, using the DNA-mediated gene transfer technique, the cDNA of SEQ ID NO: 127 may be expressed such that the protein of SEQ ID NO: 368 or a portion thereof is overexpressed in these cell lines.
The sequence or part thereof can be introduced into a cell line. The effect of this overexpression on N-glycosylation of proteins can then be studied using immunoblotting or western blotting of glycoproteins <Makishima et al. (1997) Genes Cells
2: 129-141; Silberstein et al., (1995) J. Cell. Biol. 131: 371-383; Hong.
Et al. (2000) Developmental Biology 220>. The activity of the protein of the invention, or a portion thereof, in cell apoptosis can be assayed using any of the assays known to those of skill in the art, including those described in Nakashima et al., (1993), supra. One object of the invention is compositions and methods for targeting a heterologous polypeptide to the endoplasmic reticulum by recombinantly or chemically fusing a fragment of the protein of the invention to the heterologous polypeptide. A preferred fragment is a protein of the invention or a portion thereof that contains a targeting signal to the endoplasmic reticulum, for which a Pi
doux AL, Armstrong EMBO J 1992 Apr; 11 (4): 1583-91; Munro S, Pelham HR C
ell 1987 Mar 13; 48 (5): 899-907; Pelham HR Trends Biochem Sci 1990 Dec
15 (12): 483-6. In another embodiment, the invention provides for stimulating the initiation of apoptosis of a cell,
Compositions and methods using the proteins of the invention or portions thereof. In a preferred embodiment, an effective amount of a pro-apoptotic protein of the invention or a portion thereof to stimulate apoptosis is added to an in vitro culture of mammalian or plant cells. In another preferred embodiment, the transgenic cDNA sequence of SEQ ID NO: 127, or a portion thereof, is capable of expressing the disclosed protein of the invention, or a portion thereof, at high levels relative to normal, whenever and where desired. It can be used to create animal or plant cells. Methods for making transgenic cells capable of modulating transgene expression when desired are known in the art. Increasing the expression level of the proteins of the invention to stimulate programmed cell death makes certain cell types unnecessary for certain events, such as infection, transformation, end of the production process, etc. It is useful in some cases.

Furthermore, the present invention provides immunodeficiency syndromes (including AIDS), type I diabetes, pathogenicity.
Infection, cardiovascular and nerve damage, alopecia, aging, Alzheimer's disease, Parkin
Son's disease, Huntington's disease, dystonia, Leber's hereditary visual neuropathy, sperm
Schizophrenia, "Mitochondrial encephalopathy, lactic acidosis, and stroke" (MELAS)
And "Myoclonic tencan clonic muscular spasm crude red fiber syndrome" (MERRF)
Abnormal cell proliferation and / or disorders including, but not limited to, muscle degenerative disorders such as
, Diagnosis and / or prevention of disorders characterized by programmed cell death
It relates to compositions or methods of using the proteins of the invention in therapy. For diagnostic purposes
The expression of the proteins of the invention is described in Northern Blot, RT,
-Can be studied using PCR, immunoblotting, expression and ratio in control individuals
Compared. Disorders requiring reduced cell proliferation and / or increased apoptosis
For the purpose of prevention and / or treatment of
Alternatively, it can be enhanced using any of the gene therapies known to those of skill in the art.
. Prevention of disorders that require increased cell proliferation and / or decreased apoptosis
For therapeutic and / or therapeutic purposes, the suppression of endogenous expression of the proteins of the invention may be
Professionals that include a triple helix or antisense strategy as detailed in the book
This can be achieved using any of the methods known to those skilled in the art. Furthermore, antibodies against the proteins of the invention or parts thereof are known to those skilled in the art.
Any of the techniques can be used to detect endoplasmic reticulum for histological purposes. <The protein of SEQ ID NO: 284 (internal designation number: 174-38-3-0-C9-CS)> The protein of SEQ ID NO: 284 encoded by the cDNA of SEQ ID NO: 43 is a salivary gland.
Overexpressed in. 406 of the present invention, which is similar in size to fucosyltransferase.
The mino acid length protein consists of Pfa of the fucosyltransferase family from residues 70-406.
Present the m motif. Furthermore, the protein of the present invention is a Drosophila melanogaster.
D. Homologous to the putative fucosyltransferase (STR accession numbers Q9VLC1 and Q9VLC1)
. The protein of SEQ ID NO: 284 is a Brachydanio renio (EMBL accession number: AB02362
7), Schistosoma mansoni (Schistosoma mansoni) (GENPEPT accession number: AF183577-1),
Bovine (SPTREMBL accession number: Q9TQQ3), and human species (GENPEPT accession number: AJ
132772_2) alpha-1,3 fucosyltransferase (EC 2.4.1.152)
) Has homology with. Like the fucosyltransferases, the proteins of the invention are short
N-terminal cytoplasmic tail, 9-29 amino acid signal-anchor transmembrane domain, and
We present the characteristics of a type II transmembrane protein with a large C-terminal domain. It
In addition, the protein of the present invention was found to transfer alpha-1,3 fucosyl groups from residues 315-345.
Exhibits an almost perfect common motif for enzymes (Breton et al., Glycobiology 1998
; 1: 87-94). Fucosyltransferase is a fucose derivative of GDF fucose.
Galactose in the rufa 1,2 bond, and alpha 1,3-, alpha 1,4-, and
Of an enzyme that catalyzes the transfer to acetylglucosamine in the alpha and alpha 1,6-linkages
It is a family. All fucosyltransferases use the same nucleotide sugar
Therefore, its specificity probably depends on the recognition of the receptor and the type of binding formed.
Will depend on it. In human species, type II membrane proteins are found in the Golgi
, Fucosyltransferases are divided into three distinct families (Breton et al., Glyc
obiology 1998; 1: 87-94): (1) Difference between type I and type II glycans
Produces a nearly identical product because the difference is only one carbohydrate bond,
Alpha 1,2-fucosyltransferases, hFUT1 and hFUT2. hFUT1 is the ABO of red blood cells
It determines the expression of type O antigen (H antigen) in the blood group system, but hFUT2 (Se) is saliva
Determines the secretory state of its expression in (2) certain regions are alpha 1,2- and al
It exhibits similarity to the 1,6-fucosyltransferase, but another homologous protein
Alpha 1,3-fucosyltransferase that constitutes the Amily (Breton et al., Glycobiolo
gy 1998; 1: 87-94). To date, five rufa 1,3-fucosyl groups in human species
Transferases, ie hFUT3 (Lewis enzyme), hFUT4 (myeloid type), hFUT5, hF
UT6 (plasma type), and hFUT7 have been characterized. These are carbohydrate antigens
Involved in the final step of ABH sialyl-Lewis biosynthesis (de Vries et al., J Biol C
hem 1995; 270: 8712-22; Kimura et al., Biochem Biophys Res Commun 1997 8:
237: 131-7); (3) Alpha-1,6-fucosyl group transfer fermentation involved in N-glycan synthesis
Raw, hFUT8 (Miyoshi et al., Biochim Biophys Acta 1999; 1473: 9-20). Fukoshi
Cell surface glycoconjugates are involved in fertilization, embryogenesis, lymphocyte tracking, immune response and
And important roles in physiological and pathological processes such as cancer metastasis (Stau
dacher et al., Trends Glycosci Glycotechnology 1996; 8: 391-408). further
Specifically, fucosylated cell surface glycoconjugates on the apical surface of diverse epithelia are
Loli (Umesaki et al., Science 1997; 276: 964-5), E. coli (Vogeli et al., Schweiz
Arch Tierheilkd 1997; 139: 479-84) and other viruses, and HIV and other viruses (A
li et al., Infect Dis 2000; 181: 737-9), which contributes to resistance to various microorganisms.
It On the other hand, aberrant upregulation of fucosyltransferases is unique in multiple types of tumors.
It is commonly discovered and is responsible for the increased production of fucosylated glycoconjugates. So
Fucosylated glycoconjugates such as are also useful as tumor markers and (1) h
Circulates the sialyl-Lewis structure produced by FUT3,
Ca19-9 cancer antigen (Koprowski et al., Somatic Cell Genet. 197) used for diagnosis
9; 5: 957-71), and (2) its alpha 1 in hepatoma (hepatocellular carcinoma).
Alpha-fetoprotein with reduced 6,6-fucosylation (Miyoshi et al., B
iochim Biophys Acta. 1999; 1473: 9-20). On the other hand, fucosylated glycoconjugate
Aberrant production of Escherichia coli through their interaction with E and P selectins on endothelial cells
Selection by promoting extravasation of tumor cells to participate in endothelial adhesion
There is an advantage in providing economic growth (Butcher and Picker, Science 1996; 272: 60).
-6). Therefore, the modulation of fucosyltransferase activity is associated with hepatoma (Mi
Yoshi et al., Biochim Biophys Acta. 1999; 1473: 9-20), and colorectal cancer.
(Weston et al., Cancer Res. 1999; 59: 2127-35).
Can modify the tumorigenesis. Accordingly, the protein of the invention of SEQ ID NO: 284 is a sugar
Transferase, preferably hexosyl transferase, more preferably fucosyl transferase
Transferase, and more preferably, alpha 1,3-fucosyltransferase.
, Fertilization, embryogenesis, lymphocyte tracking, immune response and cancer metastasis and diversity
Involved in resistance to various microorganisms. Preferred polypeptides of the present invention are 70-40
A polypeptide containing the amino acids of SEQ ID NO: 284 from position 6 and positions 315-345.
is there. Other preferred polypeptides of the invention are bioactive as described herein.
A fragment of sequence 284, having either. The protein of the invention or
Its partial glycosyltransferase activity is (Palcic et al., Carbohydr Res 1990; 196: 133-40).
It can be assayed using any of the assays known to those of skill in the art, including those described.
. Are fucosylated compounds scarce in both therapeutic and clinical assay reagents?
There is a possibility. However, the potential for commercial and / or therapeutic purposes
The synthesis of glycosylated compounds possesses the natural properties of the saccharide subunits.
It ’s difficult. Numerous regioisomers in which different substituents of the sugar are involved in bond formation,
And the formation of different anomeric forms is possible. As a result of these problems,
Large-scale chemical synthesis of any carbohydrate is economical because of the low yield of the desired product.
Not possible due to prudentity. Glycosyltransferases like Fucosyltransferase
The enzymatic synthesis used provides an alternative to the chemical synthesis of carbohydrates. Glycosidase
Enzymatic synthesis using a glycosyltransferase, a glycosyltransferase, or a combination thereof,
It has been considered as a promising approach to product synthesis. In fact, enzyme-mediated catalysis
Provides a tremendous advantage over traditional organic synthetic pathways and produces high yields of carbohydrates.
Economically, under mild conditions in an aqueous solution, in addition to producing large amounts of unnecessary by-products
Produce without messing. Even now, however, these proteins are only available in low concentrations
Because of their propensity to be membrane-bound, such enzymes have been discovered, especially in mammals, for example.
It is difficult to isolate from a eukaryotic source such as a source. Difficult to isolate
The receptor (peptide) specificity of glycosyltransferases is poorly understood.
Absent. Therefore, recombinant glycosyltransferases containing fucosyltransferase, which can be produced in large quantities, are included.
There is a need to obtain a syltransferase. Therefore, the present invention provides glycoproteins and glycolipids.
, Or an oligosaccharide, more specifically a fucosylated compound
Method of using the protein of the present invention or a part thereof for the synthesis of a fluorinated compound.
And composition. If necessary, the protein of the present invention or a part thereof may be added to rice.
Using any of the methods known to those skilled in the art, including those described in national patent 5,776,772.
To remove its transmembrane domain and / or its Golgi retention signal
Can be produced as a soluble form. For example, the protein of the present invention or its
Some allow glycosylation, and more particularly fucosylation, as well as this combination.
GDP-fucose and substrate under conditions capable of catalyzing glycosylation of products
Add to sample containing compound. In a preferred embodiment, the protein of the invention
The enzymatic reaction carried out according to US Pat. Nos. 5,409,817 and 5,374,541 is described in US Pat.
A series of other chemicals aimed at the synthesis of complex glycosylated compounds, such as
And / or part of an enzymatic reaction. Another preference for implementing the method on a commercial scale
In a preferred embodiment, it is advantageous to immobilize the glycosyltransferase on a support.
Will. This immobilization is due to removal of enzyme from the batch product and subsequent re-enzyme.
Easy to use. Immobilization of glycosyltransferases can be achieved, for example, from transferases.
Of the membrane-binding domain of the
Can be achieved by Other methods of immobilization are also available to those of skill in the art,
It will be understood that this is explained in the published literature. In the preferred embodiment
The present invention relates to glycosylated compounds, preferably fucosylated proteins of the present invention.
Or a process for producing a portion thereof, comprising a protein of the invention.
Cells genetically engineered to produce quality or a portion thereof
In combination with one or several other cells capable of producing donor substrates for proteins
Use together. In another preferred embodiment, the invention relates to cellular glycosyl
To produce one or more enzymes that supply the internal control of the oxidative mechanism.
Glycoproteins in engineered insect, plant, or animal cells
Processes and compositions for controlling silation. Preferably, the invention is
, The fucosyltransferase of the present invention alone or in combination with other glycosyltransferases
Chinese hamster ovaries (C
HO) cell lines. This addendum fucosyltransferase modifies glycosylation machinery
And sugar sugars with a carbohydrate structure more similar to naturally occurring human glycoproteins
Produces quality. For carrying out the above process as well as for producing the above composition
The method is performed using methods known to those skilled in the art and described in US Pat. No. 5,047,335.
To be done. Another embodiment of the invention is the present method for detecting fucosylated conjugates.
The present invention relates to compositions and methods that use pak or a portion thereof. In a preferred embodiment
SEQ ID NO: 284 or a portion thereof to obtain a reagent such as an antibody
Used for. These reagents are used in radioimmunoassays, competitive binding assays, waste
Tan blot analysis, enzyme-linked immunosorbent assay (ELISA), immunochemistry, or
Other techniques known to those skilled in the art can be used (Palcic et al., Carbohydr Res 1990; 196.
: 133-40). In a preferred embodiment, the anti-antibody produced against the protein of the invention is
The body specifically visualizes salivary glands or gastrointestinal tissues (and cells derived from those tissues)
Supply the tools to do. This is the origin or origin of the cell, for example a cancer cell.
Identification for identification and identification, for example for histological slide evaluation
Useful for numerous applications, including facilitating the identification of cells and tissues of
Can be Such an assay may also detect salivary gland, prostate, liver, and gastrointestinal tract disorders and diseases.
To diagnose a variety of disorders including, but not limited to, neoplasias such as pancreatic cancer and pancreatic cancer.
Can be used. Many, including tumor tissues or other biological samples such as semen or plasma
Various types of samples can be assayed. The invention further provides a protein of the invention or
Obtained using some of the processes described herein, in part
A glycosylated compound, preferably a fucosylated compound. Such a change
Compounds include cancer, cystic fibrosis, ulcers, inflammation and arthritis, impaired fertility, and
Including but not limited to immune disorders, including autoimmune diseases such as hypothyroidism.
It can be used for the diagnosis, prevention and / or treatment of disorders that do not occur. These conditions are
Infections with active infections anywhere on the body, such as uveitis and salpingitis;
Septic shock, disseminated intravascular coagulation, and / or adult respiratory distress syndrome
Complications of infection, including; acute or chronic inflammation due to antigen, antibody and / or complement deposition
Disease; arthritis, cholangitis, colitis, encephalitis, endocarditis, glomerulonephritis, hepatitis, myocarditis, pancreas
It includes inflammatory conditions including inflammation, epicarditis, reperfusion injury and vasculitis. Immune diseases
T-cells such as acute or delayed hypersensitivity, graft rejection, and graft-versus-host disease
And / or macrophage-related conditions; type I diabetes and multiple sclerosis
Including, but not limited to, autoimmune diseases. In the preferred embodiment
, These glycosylated compounds or their derivatives are
Trap pathogens and, thus, pathogens and endogenous ligands
Can be used as an agent that reduces the binding of For example, such compounds are
Prevents the adhesion of cancer cells to the inner wall of blood vessels or the aggregation between cancer cells and platelets.
And / or inhibiting cancer metastasis to neutrophil vascular endothelial cells
Can be used to prevent and / or inhibit the attachment of Other obstacles are green
Includes infections in which recognition of cosylated products is essential for the development of the infection. like that
Infections include those caused by viruses such as H. pylori, E. coli and HIV.
, But not limited to it. In a preferred embodiment, such compounds, preferably
Is an oligosaccharide used as a gram-positive antibiotic and antiseptic (US Pat. No. 4,
851,338 and 4,665,060). The present invention further provides glycosylated compounds, preferably
Or the recognition of fucosylated compounds is impaired or needs to be impaired
A protein of the invention for the diagnosis, prevention and / or treatment of some disorders;
Relates to methods and compositions using a portion thereof. For diagnostic purposes, the tamper of the present invention
Expression of proteins can be determined by Northern blot, RT-PCR, immunoblotting, immunoblotting as described herein.
Can be used to study and compared to expression in control individuals. Prevention
For therapeutic purposes and / or therapeutic purposes, inhibition of endogenous expression of the present invention is described herein.
Antisense or triple-helix method and protein activity antago
Use any of the nysts to reduce the production of organism-harmful glycosylated compounds
Can be used for In another embodiment, the diverse substance is a tamper of SEQ ID NO: 284.
Treatment, attenuation and / or treatment of abnormal conditions associated with disproportionate amounts and / or activity
Or it can be used for prevention. Such substances include agonists and antagonists
, Nucleic acids and compounds such as antibodies, but are not limited thereto. In particular, the present invention
Proteins or parts thereof are glycosylated for mechanistic and clinical applications.
Inhibitors of transferases, particularly fucosyltransferase inhibitors
Can be used for development (Taylor, Curr Opin Struc Biol 1996; 6: 830-7; Colman
, Pure Appl Chem 1995; 67: 1683-8; Bamford, Enz Inhib 1995; 10: 1-16
Khan & Matta. Glycoconjugates, compositions, structures and functions (In Glycoconjugates,
Composition, Structure, and Function) pp361-378.eds., Allen, HJ &
Kisailus, EC Marcel Dekker, Inc. New York, 1992; Thorne-Tjomsland
Et al., Transplantation 2000; 69: 806-8; Basset et al., Scand J Immunol 2000.
51: 307-11). Such substances are used in species containing disorders caused by certain types of neoplastic tumors.
It can be used for various therapeutic and prophylactic treatments. For example, the tongue of SEQ ID NO: 284
Substances that target the protein include, but are not limited to, salivary glands, prostate, and liver.
It can be administered to treat patients suffering from diseases of the fetal tract and pancreatic cancer. There
Such substances are used for the treatment of infectious diseases in order to induce resistance to various microorganisms.
Can be used for medical treatment, attenuation and / or prevention. <The protein of SEQ ID NO: 292 (including
Part designation number 181-10-1-0-D10-CS)> The protein of SEQ ID NO: 292 is the cDNA of SEQ ID NO: 51.
Coded by Therefore, the poly of SEQ ID NO: 292 described throughout this application
All of the peptide features and uses are also included in clone 181-10-1-0-D10-CS
It will be understood that it relates to the polypeptide encoded by the nucleic acid
. Furthermore, all of the features and uses of the nucleic acid of SEQ ID NO: 51 described throughout this application
Is also understood to relate to the nucleic acid contained in clone 181-10-1-0-D10-CS.
Will The protein of SEQ ID NO: 292 is a library constructed from fetal liver.
Was identified in the cDNA from Lee. Tissue distribution analysis using a database
It was shown that the mRNA encoding the protein is found mainly in fetal kidney and fetal liver.
did. The protein of SEQ ID NO: 292 is incorporated by reference in its entire publication.
Sequences from the proteins described in International Patent Application Publication WO 9906553-A2
It appears to be a polymorphic variant of the human secretory protein numbered 197 (92% identity). It
In addition, the protein of SEQ ID NO: 292 is a mouse macrophage asialoglycoprotein.
Binding protein (M-ASGP-BP, 36% identity), mouse natural killer (NK)
Cell surface proteins P1 40 (NKR-, P1.9, 34%) and EP 773289-A2-derived human diers
It is homologous to the C-type lectin domain of the sialoglycoprotein receptor L-H2 (27%).
Accordingly, the present invention provides nucleic acid and amino acid sequences for lectin-like proteins, and
, The use of these sequences in the diagnosis, research, prevention and treatment of diseases.
The protein of SEQ ID NO: 292 consists of 111 amino acids. Amino acid alignment and hydrophobicity
From the sex plot, the predicted signal peptide sequence consisting of 12 to 24 residues and 5 to 25 residues are shown.
It has one predicted transmembrane domain consisting of a group. Therefore, one embodiment of the present invention
The condition is a polypeptide containing a signal peptide or a transmembrane domain. I
Several distinct protein families first in several animal lectins
And functions as a calcium-dependent carbohydrate recognition domain.
Shares possible conserved domains (Drickamer K., J. Biol. Chem., 263:
9557-9560, 1988, the entire disclosure of which is incorporated herein by reference.
). Known as C-type lectin domain (CTL) or carbohydrate recognition domain (CRD)
This domain, which consists of approximately 110-130 residues. Perfectly preserved, two giz
There are four cysteines involved in Ruffido binding. CTL domain explained
Several categories of proteins have been discovered. M-ASGP-BP and NKR-P1
Are both type II membrane proteins. CTL domain is located at the C-terminus
Type II membrane proteins include: 1) also known as liver lectin,
Asialoglycoprotein receptor (ASGPR). ASGPR is a C-terminal in the carbohydrate part
Mediates endocytosis of plasma glycoproteins with terminal sialic acid residues removed
To do. 2) NK cells, and some subsets of T cells: NKG2, NKR-P1, Ly-
49, CD69 and B cells: CD72, several tampers expressed on the surface of LyB-2
Quality. The CTL domain in these proteins is distant from other CTL domains.
However, it is not clear if they all bind carbohydrates. M-AS
GP-BP is a lectin-like molecule expressed on the surface of activated macrophages.
Specific for terminal D-galactose and N-acetyl-D-galactosamine units
(Oda S et al., J. Biochem., 104: 600-605, 1988, the disclosure of which is the entire
It is incorporated as a reference in the detailed book). Experimental results show that M-ASGP-BP is a tumoricidal
It is suggested to be involved in the interaction between rophage and tumor cells. ASGPR is the liver
It is a membrane protein that is specifically expressed by cells. Its function is in the liver
Incorporation of asialoglycoprotein into serum for solution. Partially deguded
Lycosylated plasma glycoproteins circulate through a receptor-mediated process
Efficiently and specifically removed from. In mammals, desialylated (galactosyl powder)
End) ASGPR specific for glycoprotein is exclusively expressed in hepatocytes. This thin
After the ligand binds to the cell surface receptor, the receptor-ligand complex forms a series of small membranes.
Vesicles and tubules within acidic preparative organelles where the receptor and ligand dissociate
Transferred and transported (Spiess M et al., J. Biol. Chem., 260: 1979-1982, 198).
5, the full text of which is incorporated herein by reference). Of AGPR expression
Decrease in response to liver conditions such as cirrhosis, liver cancer and regenerating liver
Reported (Stadalnik et al., J. Nucl. Med., 26: 1233-1242, 1985,
The disclosure is incorporated herein by reference in its entirety). ASGPR itself in serum
Have been reported to exist (Katsugi et al., Alcohol Metabolism and L.
iver, 12: 65-68, 1992, the disclosure of which is incorporated herein by reference in its entirety.
, Which is an obvious cause for pursuing research on the measurement of serum ASGPR.
It was. Furthermore, published results show that labeled compounds that bind ASGPR are
(Kudo et al., Japan Assoc.
of Gastrointest. Pathology., 89: 1349-1359, 1992, the disclosure of which is the entire text of this application.
Incorporated in the description as a reference). NK cells are the third major lymphocyte
Make up a group. They kill cells infected with a variety of tumors and viruses
It has the unique ability to cause autologous transplant rejection. These characteristics are
, Play a major role in the regulation of innate immune responses, and broadly immune function.
And enable. Members of the NKR-P1 family are targets found on the surface of NK cells.
Ip II transmembrane C-type lectin receptor and a subset of T lymphocytes (NK T cells)
is there. Furthermore, a subset of NKR-P1 molecules are present on the surface of peripheral blood monocytes and dendritic cells.
Have been identified (Poggi A et al., Eur. J. Immunol., 27: 2965-2970, 1997,
The disclosure of which is incorporated herein by reference in its entirety). Mainly liver
And NKR-P1 that accumulates in bone marrow ⁺ T cell deficiency is associated with insulin-dependent diabetes mellitus (IDDM
, Tori M et al., Transplantation, 70: 32-38, 2000, the entire disclosure of which is hereby incorporated by reference.
Incorporated by reference in the detailed text) and multiple sclerosis (Poggi A et al., J. Imm.
unol., 162: 4349-4354, 1999, the disclosure of which is incorporated herein by reference in its entirety.
Are associated with susceptibility to a number of diseases, including NKR-P1 receptor
Was shown to activate NK cell cytotoxicity coupled with the release of interferon- (
(IFN- (, Brown MG., Immunol Rev., 155: 55-75, 1997, that
The disclosure is incorporated herein by reference in its entirety). However, receiving NKR-P1
Well-characterized to control the specificity of the Ly-49 molecular family structurally related to the organism
Unlike the assigned MHC class I ligand, the cognate ligand of the NKR-P1 molecule is still
Not identified. Interestingly, a subset of NKR-P1 molecules-NKR-P1B-
It has been reported to inhibit NK cell activation (Carlyle et al., J. Immunol., 16
2: 5917-5923, 1999, the disclosure of which is incorporated herein by reference in its entirety.
). Based on structure and chemical homology, the protein of SEQ ID NO: 292 has
Ligand binding can be calcium-dependent, C-type leptin-like, type II membrane proteins
It was characterized as a pak quality. The protein of SEQ ID NO: 292 or its fragment
Can provide the basis for clinical diagnosis of diseases associated with its induction and / or suppression.
Wear. This protein, its fragment, or its antagonist / inhibition
Bitter can cause tumors, viral infections, inflammation or immune damage, organ transplants, bacterial infections, autologous
Useful for diagnosing and treating conditions related to autoimmunity, liver dysfunction and regenerating liver
obtain. In addition, the protein of SEQ ID NO: 292 or a fragment thereof is normal and
And cytokines such as IFN and IL-12 and IL-4 in diseased cells
And used as reagents to analyze the regulation of gene expression by growth and transcription factors.
Can be used. The protein of SEQ ID NO: 292 is ASPRG, M-ASGP-BP and NKR-P1.
It has homology to the CTL domain of the molecule. Sequences in membrane bound or soluble form
No. 292 protein promotes transendothelial migration, anti-inflammatory activity, tumor suppressor activity, etc.
For cytokine receptor activity, cell proliferation / differentiation activity, T cell activation activity,
It may have tissue growth regulating activity, receptor / ligand activity, signaling activity. Therefore
, SEQ ID NO: 292, or a fragment thereof,
Rheumatism, Graves' disease, systemic lupus erythematosus, Wegener's granulomatosis, monkey
Kodosis, polyarthritis, pemphigus, erythema, polymorphism, Sjogren's syndrome, inflammation
Enteritis, autoimmune encephalitis, myasthenia gravis, keratitis, scleritis, lupus, nephritis, are
Autoimmune disorders such as luge encephalomyelitis; leukemia, lymphoma, (Hodgkin and non-
Hodgkin's sarcoma, melanoma, line cancer, solid tissue carcinoma, hypoxic tumor, mouth, pharynx
, Larynx, and lung squamous cell carcinoma, cervical cancer, and bladder cancer
, Head and neck cancer, nervous system cancer, papilloma, atherosclerosis, blood vessels
Proliferative disorders including various forms of cancer such as benign lesions such as plaque formation; viral infections,
HBV, HCV and HIV infection, and other viral and pathogen-induced
It can be used to diagnose and treat diseases such as, but not limited to, infections.
Wear. The protein of SEQ ID NO: 292 or fragments thereof may also cause inflammation or immunity.
Epidemics (eg rheumatoid arthritis and osteoarthritis and AIDS), allergies, liver cirrhosis
And liver poisons, as well as genetic diseases, chronic diseases and NK, NKT, macrophages
To treat infection-related conditions associated with decreased monocyte and dendritic cell function
Can be used for In another embodiment of this invention the protein of SEQ ID NO: 292
Inhibitors have demonstrated multiple sclerosis, IDMM, graft-versus-host disease (GVH) and transplant organ rejection.
It can be used to treat conditions such as extinction. Other embodiments have entered the portal vein through the intestine
To treat and / or prevent bacterial infections in the liver caused by bacterial antigens
Regarding the method. In this embodiment, the protein of SEQ ID NO: 292 is an endotoxin.
It can be used to counter the effects of lipopolysaccharide (LPS). Another embodiment of the invention is
Aims to suppress NK cells activated by bacterial superantigens or LPS
Use of the protein of SEQ ID NO: 292 or a fragment thereof, which is
It assists in the treatment of vascular endothelial injury found in the pathophysiology of Saki disease. Tampa with SEQ ID NO: 292
The appearance of autoantibodies to the cytoplasm is associated with cirrhosis and fatal refractory hepatitis, and
An indicator of autoimmune hepatitis (AIH), which can cause biliary cirrhosis
Can be used as Encodes the protein of SEQ ID NO: 292 or a fragment thereof
The nucleic acid sequence that is used can be used to produce a secreted form of the protein. Those
Can also be used in the development of diagnostic and therapeutic products. Therefore, recombinant soluble
Derivatives of the present invention can be prepared by, for example, ELISA, Western blotting, etc.
It can be used to detect and measure antibodies specific to proteins. This diagnoses AIH,
And allow it to be distinguished from other diseases. In another embodiment of the invention,
The protein of column number 292, or a fragment or derivative thereof, may also be
Analysis and purification of sialoglycoprotein and asialoglycoprotein tamper into hepatocytes
Protein uptake or inhibition of viral and other protein entry
Can be used in drug development.

Other embodiments of the invention relate to polypeptides comprising the protein of SEQ ID NO: 292 or fragments thereof, and polynucleotides comprising the protein of SEQ ID NO: 292 or fragments thereof. In another aspect, the protein of SEQ ID NO: 292 or a fragment thereof can be used to identify a particular molecule such as an agonist, antagonist, or inhibitor to which it binds. In another aspect, the invention relates to methods of identifying agonists and antagonists / inhibitors of the protein of SEQ ID NO: 292, and methods of treating conditions associated with the proteins of the invention or with the imbalance of identified compounds. In yet another aspect, the invention provides
It relates to a diagnostic assay for detecting a disease associated with inappropriate levels or activity of the protein of SEQ ID NO: 292. Another embodiment of the present invention relates to a method for measuring the amount of protein of SEQ ID NO: 292 in serum. Other embodiments relate to the use of labeled compounds that bind to the protein of SEQ ID NO: 292 and can be used as convenient indicators of liver function, NK cell activity, or the like. Embodiments of the invention relate to methods of using the proteins of the invention or portions thereof to identify and / or quantify other ligands that may interact with the protein of SEQ ID NO: 292. The protein of SEQ ID NO: 292 or fragments thereof may be included in a pharmaceutical formulation for the treatment of cancer, or for the prevention / treatment of other disorders associated with altered expression of the proteins of the invention (see above). In a preferred embodiment of the invention, the protein of the invention or a part thereof comprises cytokines and IL-1.
, IL-2, IL-12, IFN-γ and other related molecules. The protein of SEQ ID NO: 292 has also been shown to inject the protein intraperitoneally, intravenously, subcutaneously or directly in diseased tissues such as autoimmunity, inflammation, pathogen-mediated infection, liver toxicity, autologous transplant rejection, GVH, etc. It can be used to correct defects in in vivo models of disease, as well as tumor models. DNA encoding the protein of SEQ ID NO: 292 or fragments thereof can be used in diagnostic assays for conditions / diseases associated with upregulation or downregulation of protein expression of the invention (see above). The diagnostic assay is for the presence or absence of protein expression,
And is useful for distinguishing overexpression and monitoring regulation of protein levels during therapeutic intervention. DNA has also been incorporated into effective eukaryotic expression vectors to treat the above conditions, including tumors, and / or correct diseased or gene-induced defects in any of the above proteins, including the proteins of the invention. Specific tissues, organs, or cell populations can be targeted directly in the gene therapy of interest. DNA has also been used to design antisense sequences and ribozymes to modify gene expression in NK, NKT, macrophages, monocytes and dendritic cells, and IL-1, IL-2, IL-4, IL-. 12 and IFN-γ can be administered to influence the expression of cytokines. In vivo delivery of the genetic construct into a subject may affect expression of the protein of SEQ ID NO: 292, or expression and / or activity of other proteins such as cytokines, growth factors, their receptors and / or tumor antigens. Points can be developed that target specific cell types, such as modulating tumors. DNA also identifies IFN in unknown upstream sequences (eg, promoters and regulatory elements), and in normal and diseased cells by standard methods.
And other cytokines, and can be used to study the regulation of gene expression by growth and transcription factors. The hybridization probe is D, which encodes the protein of SEQ ID NO: 292 (or its similar molecule) in a biological sample.
It is useful for detecting NA, as well as mapping naturally occurring genomic sequences to specific chromosomes / regions. DNA has created and / or based on vaccine, knockout, transgene technology, in vivo animal models of disease, including infection, tumors, autoimmune conditions, GVH, autograft rejection, and susceptibility or resistance to liver venom.
Or it can be used to treat.

Antibodies to the protein of SEQ ID NO: 292 are useful in diagnosing conditions and diseases associated with expression of the proteins of the invention and quantifying the protein (eg, patient monitoring assays during therapeutic intervention). Antibodies specific for proteins can include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments produced by Fab expression libraries. Neutralizing antibodies are particularly preferred for diagnostic and therapeutic methods. The diagnostic assay for the protein of the present invention is a method utilizing an antibody and a label for detecting the protein of SEQ ID NO: 292 in human body fluids or cells or tissue extracts, and the detection or measurement of the antibody against the protein of SEQ ID NO: 292. Including the method. The protein of SEQ ID NO: 292 and its catalytic or immunogenic fragments, or oligopeptides thereof, can be used to screen therapeutic compounds in a variety of drug screening techniques, including high throughput. Methods that can be used to quantify the expression of the nucleotides or proteins of the invention include polymerase chain reaction (PCR), RT-PCR, RNAse protection, Northern blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay. Method (RIA), fluorescence-activated cell sorting (FA
CS), immunoprecipitation, and chromatography. Thus, the protein of SEQ ID NO: 292 or fragments thereof can be used for purification or enrichment of carbohydrate containing proteins. In such embodiments, the lectins of the invention are placed in contact with carbohydrate-containing proteins under conditions that promote specific binding. The lectin of the present invention may be immobilized on a solid support. After binding, the specifically bound proteins are dissociated using appropriate salts or other conditions. The protein of SEQ ID NO: 292 or a fragment thereof also interacts with tumoricidal macrophages and tumor cells, NK cell activity, treatment of bacterial infections caused by bacterial antigens being introduced from the intestine, or of bacterial LPS. It can be used to control any of the above activities, including counteracting the effects. Accordingly, the present invention provides at least 5, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 6 having the desired biological activity to treat or ameliorate a condition in an individual.
Includes uses of the protein or fragment of SEQ ID NO: 292, which comprises 0, 75, 100, 150 or 200 contiguous amino acids, or fragments thereof. For example, the condition can be an abnormality of any of those described above or of any of the functions listed above. In such embodiments, the protein of SEQ ID NO: 292 or fragment thereof is administered to an individual who desires to increase or decrease the activity of the protein of SEQ ID NO: 292. The protein of SEQ ID NO: 292 or a fragment thereof may be administered directly to an individual, or the nucleic acid encoding the protein of SEQ ID NO: 292 or a fragment thereof may be administered to an individual. Alternatively, SEQ ID NO: 29
An agent that increases the protein activity of 2 may be administered to the individual. Such agents are prepared by contacting the protein of SEQ ID NO: 292, or a cell, or a preparation containing the protein of SEQ ID NO: 292, with a test agent and analyzing whether the test agent increases protein activity. Can be identified. For example, the test agent can be a compound or polypeptide or peptide.

Alternatively, the protein activity of SEQ ID NO: 292 may be reduced by administering to the individual an agent that interferes with such activity. An agent that interferes with the activity of the protein of SEQ ID NO: 292 is a protein of SEQ ID NO: 292, or a cell, or a preparation containing the protein of SEQ ID NO: 292 is contacted with a test agent to determine whether the test agent increases protein activity. It can be identified by analyzing whether or not. For example, the test agent can be a compound, polypeptide or peptide, antibody, or nucleic acid such as an antisense nucleic acid or a triple helix forming nucleic acid. In one embodiment, the invention selectively identifies the source of the sample, such as fetal liver or fetal kidney, based on the protein level of SEQ ID NO: 292 in the sample, or, alternatively, two or more Methods and compositions using the protein of the invention or a portion thereof as a marker protein for identifying the source from which the sample may be derived. For example, the protein of SEQ ID NO: 292 or a fragment thereof, using techniques known to those of skill in the art, including those described herein,
It can be used to produce antibodies. Such antibodies may then identify or identify tissue of unknown origin, such as differentiated tumor tissue that has metastasized to forensic samples, different body sites, etc.
It can be used to classify different tissue types in a tissue cross section using immunochemistry. In such methods, a sample is contacted with a detectably labeled antibody under conditions that promote antibody binding. The level of antibody bound to the test sample is measured and compared to the level of binding to control cells from fetal liver or kidney, or to control cells from tissues other than fetal liver or kidney, to determine whether the test sample has fetal liver or Determine if it is from fetal kidney. Alternatively, the protein level of SEQ ID NO: 292 in the test sample may be measured by determining the RNA level encoding the protein of SEQ ID NO: 292 in the test sample. RNA levels can be measured using nucleic acid arrays or by methods such as in situ hybridization, Northern blot, dot blot or other methods known to those of skill in the art. Optionally, an amplification reaction such as a PCR reaction may be performed before analyzing the nucleic acid sample. The test sample is derived from fetal liver or kidney by comparing the RNA level in the test sample with the RNA level in control cells from fetal liver or kidney or from tissues other than fetal liver or kidney. To determine.

In another embodiment, antibodies to the protein of the invention or portions thereof are used to detect, enrich, or purify cells expressing the protein of SEQ ID NO: 292, using methods including methods known to those of skill in the art. Can be used for For example, an antibody to the protein of SEQ ID NO: 292 or a fragment thereof can be immobilized on a solid support such as a chromatography matrix. A preparation comprising cells expressing the protein of SEQ ID NO: 292 is arranged to contact the antibody under conditions that promote binding to the antibody. The cells are released from the support by washing the support and then contacting the support with an agent that dissociates the cells from the antibody. In another embodiment of the invention, the protein of SEQ ID NO: 292 or a fragment thereof can be used to diagnose a disease associated with modified expression of the protein of SEQ ID NO: 292. In such techniques, techniques known to those of skill in the art are used to measure protein levels of SEQ ID NO: 292 in diseased individuals. The protein level of SEQ ID NO: 292 in a diseased individual is compared to the level in a healthy individual to determine whether the individual has a level of the protein of SEQ ID NO: 292 associated with the disease. <Protein of SEQ ID NO: 408 (Internal Designation Number 179-14-2-0-F11-CS)> SEQ ID NO: 409 of SEQ ID NO: 409 encoded by the cDNA of SEQ ID NO: 168, referred to herein as PNMT A The protein is found in fetal kidney and fetal brain. PNMT A is a human phosphotidyl ethanolamine N-methyltransferase (PN
MT) polymorphic variant (SPTREMBLNEW SPTREMBL SWISSPROT accession number Q9UHY6). PNMT A differs from the sequence of PNMT (STR Accession No. Q9UHY6) by two amino acid residues. Position 95 contains an isoleucine residue (I) that replaces the valine residue (V) and position 130 contains a valine residue (V) that replaces the glutamine residue (G). PNMT A presents four candidate transmembrane segments at positions 50-70, 83-103, 131-151 and 196-1216. Catecholamine neurotransmitters <e.g. Dopamine, noradrenaline (norepinephrine), adrenaline (epinephrine) are synthesized from tyrosine through dopa, dopamine and noradrenaline to adrenaline in catecholaminergic neurons. Four enzymes are involved in the biosynthesis of adrenaline: (1) tyrosine 3-mono-oxygenase (tyrosine hydroxylase, TH); (2
) Aromatic L-amino acid decarboxylase (AADC, or dopa decarboxylase, DCC); (3)
Dopamine beta-mono-oxygenase (dopamine beta-hydroxylase,
DBH); and (4) noradrenaline N-methyltransferase (phenylethanolamine N-methyltransferase, PNMT) (Nagatsu, Neurosci. Res. 12: 315-345).
(1991)). PNMT, the final enzyme in the adrenaline biosynthetic pathway, catalyzes the production of adrenaline from noradrenaline using S-adenosyl-L-methionine as the methyl group donor. For this reason, PNMT serves as a convenient marker for epinephrine (adrenergic) producing tissues and cells. Studies by Kennedy and coworkers have shown that PNMT is widely distributed in human tissues including heart and kidney (Kennedy et al., J. Clin. Invest. 95: 2896-2902 (1995)).

In some pheochromocytomas, the tumor contains and secretes large amounts of adrenaline compared to normal adrenal medulla. In a case / control study, Isobe et al. Showed that adrenergic pheochromocytoma expressed significantly higher amounts of PNMT mRNA compared to normal adrenal medulla (Isobe et al., J. Urol. 163: 357-362
(2000)). Furthermore, PNMT immunoreactivity is detected only in adrenergic tumors. The C1 region, located in the rostral, ventral, and lateral medulla, contains primarily adrenergic neurons. These neurons are the tonic vasomotor centers of the brain. Burke et al., PN in axon terminals and cell bodies of neurons from the medulla of Alzheimer's disease patients.
It was demonstrated that there was a change in the enzymatic activity of MT. They also showed that the PNMT protein is reduced in the axon terminals of the brains of Alzheimer's patients, and this reduction in PNMT is likely due to degenerative degeneration of epinephrine neurons (Burke et al., Ann. Neu.
rol. 22: 278-280 (1987)). In the case of progressive Alzheimer's disease, Burke et al. Showed that the accumulation of PMNMT in the perinuclear region was due to reduced transport of this enzyme to the axon terminals (Burke et al., J. Am. Geriatr. Soc. 38: 1275-1282 (1990)). Neurons, including PNMT, have cell bodies in the brainstem region of the rat brain and send projections primarily into other brainstem regions such as the hypothalamus and spinal cord. These neurons are
It can be pharmacologically affected by a wide variety of drugs. PNMT inhibitors currently represent the only means of pharmacologically modifying adrenergic neurons without affecting noradrenergic or dopamine neurons in the brain. Deoxycorticosterone acetate (DOCA salt) hypertensive rats and spontaneously hypertensive rats (SHR)
Experiments carried out at PNMT inhibitors have shown that they reduce blood pressure (Go
ldstein et al., Life Sci 30: 1951-1957 (1982); Lyang et al., Res. Commun. Chem.
Pathol. Pharmacol. 46: 319-329 (1984); Chatelain et al., J. Pharmacol. Exp.
. Ther. 252: 117-125 (1990)). Molecules and compounds that affect adrenergic neurons may also have applications in the treatment of psychosis and neuroendocrine dysfunction. One embodiment of the invention provides a polypeptide comprising the sequence of PNMT A.
Other polypeptides of the invention include 50-70, 83-103, 131-151 and / or
Included is a polypeptide comprising the amino acids of SEQ ID NO: 409 from positions 196-216. PNMT A
Bioactive fragments of proteins are also included in the invention. A "bioactive fragment" is defined as a peptide or polypeptide fragment of PNMT A that has at least one of the biological functions of PNMT A protein (eg, the ability to catalyze adrenaline formation). In a preferred embodiment, the bioactive fragment of PNMT A is at least one amino acid substitution that distinguishes PNMT A from PNMT (ie, position 95 is isoleucine residue (I) replacing valine residue (V); and / Or position 130 contains a valine residue (V) which replaces the glutamine residue (G). In one embodiment, the PNMT A polypeptide of the invention is clone 179-1462-0-F11-CS.
Coded by

Accordingly, one embodiment of the present invention provides enzyme components of the adrenaline synthesis pathway and methods of producing adrenaline according to methods known to those of skill in the art. These methods use PNMT A, or biologically active fragments thereof, in known synthetic routes.
Replace with PNMT enzyme. The present invention also provides variants of the protein of SEQ ID NO: 409. These variants have at least about 80% amino acid sequence identity with the amino acid sequence of PMNT A, more preferably at least about 90%, and most preferably at least about 95%. Variants according to the invention also have at least one functional or structural characteristic of PNMT A, such as the ability to catalyze adrenaline formation. The present invention also provides bioactive fragments of variant proteins. Unless otherwise stated, the methods disclosed herein can be performed using PNMT A or variants thereof. Similarly, the method of the present invention can be carried out with a bioactive fragment of PNMT A, or a variant thereof, provided that said bioactive fragment contains the amino acid substitutions described above. One embodiment of the invention provides a method of using a protein of the invention, or a bioactive fragment thereof, to label (chemically or isotope) an adrenaline molecule in vitro. The labeled adrenergic molecule can then be used to reveal receptor localization in tissues by in situ hybridization experiments. The invention also provides a fusion protein or polypeptide in which PNMT A or a biologically active fragment thereof is linked to other proteins (tags) by the use of recombinant DNA molecules. The purified, enzymatically active fusion product produced was then reacted in vitro with S- as a noradrenaline precursor and methyl group donor.
Add to adenosyl-L-methionine. The enzymatic reaction is performed under conditions known to those skilled in the art (Burke et al., Proc. Soc. Exp. Biol. Med. 181: 66-70 (1986); Morimoto et al.
Endocr. J. 40: 179-183 (1993)). In this reaction, the methyl group of S-adenosyl-L-methionine was isotopically (<14c> -S-adenosyl-L- Methionine or <methyl-3H> -adenosylmethionine) or chemically labeled.

Similarly, the PNMT activity of the expressed protein in cells transfected with a cDNA encoding the protein of the present invention is <14C> -S-adenosyl-derived by cytosolic fractionation according to methods known to those skilled in the art. It can be measured by adding L-methionine and normetanephilin and incubating for 60 minutes (Morimoto et al., Ibid.).
Agonists and / or antagonists of PNMT activity can also be tested (transfected cells) expressing wild-type proteins of the invention (high throughput screening). Again, the effect of such drugs on PNMT enzyme activity is measured by the method described above. The invention further relates to methods and compositions used to modify the proteins of the invention (ie, derivatize the PNMT A protein). Post-translational modifications encompassed by the invention include, for example, N-linked or O-linked carbohydrate chains, N- or C-terminal processing, attachment of compound moieties such as polyethylene glycol to the amino acid backbone, N-linked or O-linked carbohydrate chains. It includes chemical modifications, and additions or deletions of N-terminal methionine residues as a result of prokaryotic host cell expression. Some of these modifications of the proteins of the invention may facilitate their extraction and purification in prokaryotic host systems. Post-translational modifications such as N-linked or O-linked carbohydrate chain addition can also optimize the enzymatic activity of the proteins of the invention when first produced in a prokaryotic system. Another embodiment of the invention provides an antibody directed against the protein of the invention or an immunogenic fragment thereof. The antibody of the present invention is useful for screening adrenaline-producing tissues and cells, or for affinity purification of PNMT or PNMT A. These antibodies can also be used to diagnose conditions and disorders in which PNMT A is overexpressed, such as pheochromocytoma and Alzheimer's disease. Methods of performing affinity purification methods, as well as methods of making polyclonal and monoclonal antibodies are known to those of skill in the art. Neutralizing antibodies can be used as antagonists of PNMT A in therapeutic procedures to
It can be used to treat conditions associated with TA overexpression. These disorders include, but are not limited to, hypertension, pheochromocytoma and advanced Alzheimer's disease (Goldstein et al., Life Sci 30: 1951-1957 (1982); Lyang et al., Res. Commu.
n. Chem. Pathol. Pharmacol. 46: 319-329 (1984); Chatelain et al., J. Pharma.
col. Exp. Ther. 252: 117-125 (1990); Isobe et al., J. Urol. 163: 357-362 (2.
000); Burke et al., J. Am. Geriatr. Soc. 38: 1275-1282 (1990)).

<Proteins of SEQ ID NOs: 395 and 403 (internal designation numbers 160-101-3-0-H2-CS and 160-99-4-0-E4-CS)> Encoded by the cDNAs of SEQ ID NOs: 154 and 162, respectively The 367 amino acid long proteins of SEQ ID NOs: 395 and 403 are polymorphic variants, the protein of SEQ ID NO: 395 is overexpressed in fetal brain and ovary, and the protein of 403 is overexpressed in fetal brain only. It They are both the G and H sites, respectively, as described below, the glutathione S-transferases (G and T from positions 47-122 and 206-309).
ST) domain. In addition, they also display two hydrophobic domains (from amino acid positions 258-278 and 338-358) that are characteristic of some GST proteins. The glutathione S-transferase protein (GST) produces a product (via the formation of a thioether bond with its electrophilic center) that is less reactive and more hydrophilic, and thus can be easily excreted from cells. In particular, it is a dimeric protein that catalyzes the conjugation of glutathione to a wide range of hydrophobic compounds. GST Super Family (EC 2.
5.1.18) is in fact believed to be one of the most important proteins in the detoxification of reactive electrophiles in living cells. Glutathione is a cellular tripeptide (
Gamma-glutamyl cysteinyl glycine), which is probably the most abundant amino acid derivative in higher-living cells. Cysteine, the intermediate amino acid in glutathione, has a free thiol group that can compete with nucleophilic sites on nucleotide bases in reaction with electrophiles. In cells, glutathione
Functions to conjugate xenobiotic toxic molecules in general, and electrophiles in particular, to reduce the reactivity of toxic molecules to cellular macromolecules and to target the toxic substances to subsequent metabolic and excretory pathways. . Based on amino acid sequence identity, there are at least seven major classes of GST proteins (designated alpha, kappa, mu, pie, sigma, theta and zeta). Sequence similarity between classes is rather low, ranging from 20-30%. However, a single point mutation in the H subsite region of GST is sufficient to shift substrate specificity from the pi class to the alpha class (Nuccetelli MN et al., Biochem.
Biophys.Res.Commun. 252: 184-189 (1998)). Despite relatively low sequence identity, GSTs display a high degree of structural similarity. The binding of GST molecules to glutathione is highly specific and has high affinity, but indiscriminately binds to various xenobiotics, electrophilic, and alkylating chemicals. All four major cytosolic classes of GST enzymes are found in dimeric forms with two per dimer, each independently functioning active site. The active site is the glutathione binding region (called the G site)
And a non-specific hydrophobic binding region (referred to as the H site) to accommodate electrophiles. The crystal structures of pi, mu, alpha, and theta class have been elucidated and all have similar GSH binding sites,
Its hydrophobic substrate binding site (H subsite) is mutated throughout the class (Allardyce CS et al., Biochem. J. 343: 525-531 (1999)). GST activity has been suggested to be involved in the control of multi-subunit complex assembly by shifting the equilibrium between glutathione, glutathione disulfide, the thiol group of cysteine and the disulfide bond of proteins. The GST domain is an extensive, conserved enzyme module that can be covalently or non-covalently complexed with other proteins. Control of protein assembly and folding
It may be one of the functions of GST (Koonin EV et al., Protein Sci 3: 2045-2054 (1994).
).

Cytosolic glutathione S-transferases are known to belong to four classes called alpha, mu, pie and theta. The fifth class of glutathione S-transferases are microsomal enzymes found primarily in the endoplasmic reticulum of the liver. A detailed analysis of the expression of microsomal glutathione transferase 1 in human tissues shows that it appears preferentially in the liver and pancreas. Relative expression levels in humans include liver and pancreas to kidney, prostate, colon (30-40%), heart, brain, lung, testis, ovary, small intestine (10-20%), placenta, skeletal muscle, spleen, thymus. , Peripheral blood leukocytes (1-10
%) Range. Strong expression was detected in liver in human fetal tissue, but low expression levels in lung and kidney (10-20%). No transcript was detected in fetal brain or heart ((Estonius M et al., Eur J Biochem 260: 409-13 (1999)).
Based on these observations and the fact that the enzyme is encoded by a highly conserved single copy gene, microsomal glutathione transferase 1
Are suggested to perform important essential functions in most mammalian cell types. One particular glutathione S-transferase was nevertheless identified in the mitochondrial matrix (Pemble SE et al., Biochem. J. 319: 749-754 (1996)). GST and GST-like proteins are widely dispersed in vivo. In vertebrates and cephalopods, several proteins present in the lens (crystallin) are structurally related to the alpha class GST (Chiou SH et al., Biochem. J. 309: 793-80).
0 (1995)). Furthermore, olfactory epithelial cytosol is the highest GST in extrahepatic tissues.
Shows activity. Olfactory GSTs have been discovered to catalyze glutathione conjugation of several odorant classes, including numerous unsaturated aldehydes, ketones, and epoxides, and have been proposed to have important roles in chemoreception (Ben-
Arie N. et al., Biochem. J. 292: 379-384 (1993)). Higher cells contain multiple families of GST isozymes, each with a broad, overlapping specificity for each class. Muclass GST appears to be involved in the detoxification of reactive oxygen species (cyclized o-quinones) produced by the oxidative metabolism of catecholamines. These toxins appear to be involved in nigrostriatal and mesolimbic neuropathy (Parkinson's disease and schizophrenia, respectively). The enzyme of muclass GST is expressed in the substantia nigra and has a preferential substrate specificity for cyclized o-quinones formed by catecholamine metabolism (Hansson LO et al., J. Mol. Bi.
ol. 287: 265-276 (1999), Takahashi Y. et al., J. Biol. Chem. 268: 8893-8 (19.
93)). Although most GSTs share a common substrate, there are clear differences in substrate preference among subfamilies. These enzymes evolve as cytoprotective systems against a wide variety of electrophilic compounds, including a range of xenobiotics, oxidative metabolic byproducts (oxidized lipids, DNA and catechol), and specifically metabolize some environmental carcinogens. Is known.

GST can also be used in other reactions such as peroxidase and isomerase reactions (Edw
Ards R. et al., Trends in Plant Sci. 5: 193-198 (2000), and addition of aliphatic epoxides and arene oxides to glutathione; reduction of polyol nitrates to polyols and nitrous acid by glutathione; specific isomerization. It is also known to catalyze reactions and other reactions such as disulfide exchange. Similarly, there are marked species differences in catalytic activity among various purified mammalian liver GST mixtures. Some of them catalyze the chemical stereospecific conversion of some pharmacological agents much less efficiently than others. For example, the use of recombinant human GST has led to the successful stereoconversion of 13-cis-retinoic acid to all-trans-retinoic acid (Chen H. and Juc.
hau MR Biochem.J. 336: 223-226 (1998)). The number of GST genes recognized as polymorphisms is increasing. Certain loci, especially those that result in impaired catalytic activity, may be associated with increased susceptibility to toxic compounds. Differences in gene polymorphisms and GST expression are involved in individual susceptibility to certain types of cancer (for details, see Hayes JD and Strange RC Pharmac
ology 61: 154-166 (2000)). For example, GSTM1 deficiency predisposes to head and neck cancer, especially pharyngeal cancer, especially when exposed to smoking carcinogens (Gronau S et al., L
aryngorhinootologie 79: 341-344 (2000)). On the contrary, overexpression of GST may be involved in the phenomenon of multidrug resistance to cancer chemotherapy. One class of electrophilic compounds that are substrates for glutathione S-transferases is the group of alkylating agents used in antitumor therapy. A common problem observed in modern cancer chemotherapy is the emergence of chemotherapeutic drug-resistant tumor cells, which due to their resistance do not respond adequately to anti-tumor agents. This resistance is often observed in many drugs that have no physical or mechanistic similarities to the drug substance. GST isozymes have been shown to be involved in developing drug resistance to various chemotherapeutic drugs such as adriamycin, viblastin, actinomycin D and colchicine. The resistant population of malignant cells exhibits a modified pattern of total glutathione S-transferase activity. Batis
The resistant population of MCF-7 breast cancer cells, identified by the selection of adriamycin by T. et al., J. Biol. Chem., 261: 15544-15549 (1986), is approximately 200-fold more resistant than their parental cells. Attribute a subset of cells. It was discovered that resistant cells exhibited a 45-fold increase in total glutathione S-transferase activity, the increase being a result of the appearance of isozymes not expressed in the parental cell line. An increase in glutathione S-transferase alone, which is an increase conditioned by transformation of susceptible cells with an exogenous DNA construct expressing the wild-type glutathione S-transferase coding region, can increase the resistance of the cells to antitumor agents. It was proved that Puchalski and Fahl, Pr
oc. Natl. Acad. Sci. USA, 87: 2443-2447 (1990), expression of rat 1-1, 3-3 and human P1-1 isozymes of glutathione S-transferase in COS cells , Increased its resistance to drugs. Recent studies on increased resistance of tumor cells to cytotoxic drugs or ionizing radiation have enabled the identification of a novel GST-related protein p28, which is exclusively expressed in lymphoma cells, using differential display (Kodym R. et al., J. Biol. Chem. 274: 5
11-5137 (1999)). Subcellular protein fractionation showed that p28 was localized in the cytoplasm, but that heat stress rearranged p28 into the nuclear fraction of cellular proteins. Characterization of sequence homology and similar function of p28 to other GST family members (
Rearrangement and glutathione binding capacity, especially in response to thermal stress)
Discuss that 8 is a new mammalian member of the GST superfamily.

Evidence suggests that the expression level of GST is an important factor in determining the sensitivity of cells to a wide range of toxic compounds. In humans, there are significant inter-individual differences in the expression of alpha, mu, and theta classes of GST. The mechanisms of transcriptional and post-translational regulation of the most abundant mammalian class of GSTs have been investigated. Alpha-, mu-, and pi-class biocontrol is the sex of expression-
Presents age-, tissue-, species-, and tumor-specific patterns. In addition, GS
T is regulated by a structurally diverse range of xenobiotics, and to date, T is 100
More than one compound has been identified to induce GST (Hayes JD and Pulford
DJ Crit. Rev. Biochem. Mol. Biol. 30: 445-600 (1995)). It is clear that humans may be constantly exposed to such compounds because a significant number of these compounds occur naturally and they are discovered as non-nutritive ingredients in vegetables and citrus fruits. Is. Many inducers influence the transcriptional activation of the GST gene through the antioxidant response element (ARE), xenobiotic element (XRE), GST P enhancer 1 (GPE), or glucocorticoid response element (GRE). Many compounds that induce GST are themselves substrates for these enzymes, or are metabolized (by cytochrome P-450 monooxygenase) to compounds that serve as GST substrates, which indicates that GST induction is an electrophilic substance. It suggests that it represents a part of the adaptive response mechanism to chemical stress. GST is also likely to be regulated in vivo by reactive oxygen species, a potent inducer capable of generating free radicals by redox cycling, and such regulation could be an adaptive response to oxidative stress in cells. . GST-Pie can potently and selectively suppress the activation of jun protein by its upstream kinase (JNK), and these results indicate that GST-Pai may also be a regulator of signal transduction. Suggested (Monaco R. et al., J. Prot. Chem. 18: 859-866 (1999))
. Most human tumors express significant amounts of pi-class GST (Hayes and Pulford
, Above). Therefore, GST has medical importance because it is involved in mediating drug resistance in cancer patients. In vitro measurement of GST isozymes has importance in diagnostic medicine. For example, measurement of GST pi isozymes in tissue specimens is useful in the detection and diagnostic pathology of a variety of different tumors. Furthermore, the measurement of the alpha form of GST in blood is useful for detecting and monitoring a variety of different types of liver disease (see Beckett, et al., Supra for details on the clinical application of GST measurements).

The proteins of SEQ ID NOs: 395 and 403 or parts thereof are preferably transferases that transfer alkyl or acyl groups different from methyl groups, more preferably glutathione S-transferases, especially xenobiotics and oxidants. It is thought to be a transferase involved in cell detoxification to metabolic byproducts. Preferred polypeptides of the invention are those comprising amino acids of SEQ ID NOs: 395 and 403 from positions 47-122, and 260-309. Other preferred polypeptides of the present invention are fragments of SEQ ID NOs: 395 and 403 having any of the biological activities described herein. The transferase activity of the proteins of the present invention or portions thereof is described in US Pat. Nos. 5,866,792 and 6,096,504, the disclosures of which are incorporated herein by reference in their entirety.
It can be assayed using any of the assays known to those of skill in the art, including those described for T proteins. To search for substrates, the proteins of the invention, or portions thereof, or derivatives thereof, can be used to screen compound libraries using any of a variety of drug screening techniques. The fragments used in such screens may be free in solution, attached to a solid support, cell surface, or intercellularly located. The formation of a binding complex between the protein of the invention, or a part thereof or a derivative thereof and the agent to be tested can be determined. Antagonists or inhibitors of the proteins of the invention can be produced using methods known in the art, including screening drug libraries to identify those that specifically bind the proteins of the invention. Another technique for drug screening that can be used is published international patent application WO 84/035.
As described at 64, it provides a high throughput screen for compounds with appropriate binding affinity for the proteins of the invention. The present invention relates to methods and compositions using the proteins of the invention, or portions thereof or derivatives thereof, to catalyze a GST-dependent detoxification reaction in vitro or in vivo using any of the methods known to those of skill in the art. . For example, the use of the proteins of the invention, or portions thereof, may result in toxic by-products such as those obtained in laboratory experiments,
It can be extremely useful in the treatment of dietary toxins resulting from the use of pesticides on plants used to feed animals or humans. Preferably, the protein of the present invention or a part thereof or a derivative thereof is added to the sample containing the substrate (s) under conditions capable of detoxification and catalyzing the detoxification of substrate (s). To be done. In a preferred embodiment, detoxification is performed using standard assays as described herein.

[0210] In some of the above-cited embodiments, a composition comprising a protein of the invention or a portion thereof is added to a sample as a "cocktail" with other detoxification enzymes. The advantage of using a cocktail of detoxifying enzymes is that they can detoxify a wide range of substrates without necessarily knowing the specificity of the enzyme. The use of cocktails of detoxification enzymes also
Protects samples from a wide range of unknown toxic compounds from a vast array of sources. For example, the protein of the invention or a portion thereof is added to a sample where the presence of toxic compounds is not desired. Alternatively, the protein of the invention, or a portion thereof, is bound to a chromatographic support, using techniques known in the art, alone or in combination with other detoxifying enzymes, to make an affinity chromatography column. May be. Samples containing undesired substrate are stripped of the substrate through a column. Immobilizing the protein of the invention or a portion thereof on a support is particularly advantageous for embodiments in which the method is practiced on a commercial scale. This immobilization facilitates removal of the enzyme from the batch product and subsequent reuse of the enzyme. Immobilization of the protein of the invention or a part thereof can be achieved, for example, by inserting a cellulose binding domain into the protein. One of ordinary skill in the art will appreciate that other methods of immobilization are also available and are described in the published literature. Alternatively, similar methods can be used to identify new substrates. In a preferred embodiment, the present invention uses any of the methods known to those skilled in the art,
It relates to cells and plants or animals that have been genetically engineered to express, preferably at high levels, the proteins of the invention or parts thereof. Such engineered cells, animals or plants enhance the detoxification of the compound. In a more preferred embodiment, expression of a protein of the invention or portion thereof is achieved according to US Pat. No. 5,866,79.
A technique similar to that described in 2 will be used to confer resistance to herbicides to transgenic plants. In such embodiments, the proteins of the invention may need to be modified to enhance their ability to react with a particular substrate. These modifications can provide new isoforms that are specifically effective against selected electrophilic or alkylating agents. Artificial DNA constructs encoding and expressing such modified or mutated proteins of the invention, or portions thereof, may be used in target cells to enhance their resistance to alkylating or neoplastic agents. Can be selectively delivered to. Methods for such modifications have been described (Fahl et al., US Pat. No. 6,136,605, 2000).
October 24, year). The method is based on random mutation and selection, where selection is for agents seeking enhanced activity. The mutation is preferably site-specific to the amino acid associated with the H-site of the enzyme, as it is advantageous to create new, useful isoforms of the enzyme.

In another embodiment, the present invention uses a protein of the invention, or a portion thereof, to design an artificial chemoreception-specific system as described by Ben-Arie N. et al., Supra. Compositions and methods. Such chemoreceptive systems recognize odorants, xenobiotics, pesticides, drugs, and may also be useful for chemical, cosmetic, pharmaceutical, forensic, and other analytical purposes. The design of such systems is generally based on the detailed specificity of recognition of compounds by different GST isozymes. Methods of providing analytical diagnostics based on enzyme specificity are known to those of skill in the art. In another embodiment, the invention relates to compositions and methods that employ a protein of the invention or a portion thereof as a ligand for a substrate of interest. In a preferred embodiment, the proteins of the invention, or portions thereof, can be used to identify and / or quantify substrates using techniques known to those of skill in the art, such as those described by Koonin E., supra. . In another preferred embodiment, the protein of the invention, or a portion thereof, is a two-hybrid method, an expression and purification system based on GST fusion to a heterologous protein that is already commercially available (an expression vector or plasmid encoding the fusion protein, and It can be used to improve or modify molecular biology methods based on protein-protein interactions, including but not limited to affinity purification methods). In another embodiment, the invention provides that the protein of SEQ ID NO: 403 is a marker protein for selectively identifying fetal brain, preferably the protein of SEQ ID NO: 395, as a marker protein for selective identification. Methods and compositions using the proteins of the invention or portions thereof. For example, the proteins of the invention, or portions thereof, can be used to synthesize specific antibodies using techniques known to those of skill in the art, including those described herein. Such tissue-specific antibodies can then identify tissue of unknown origin, such as differentiated tumor tissue that has metastasized to forensic samples, different body sites, etc., or use immunochemistry to identify different tissue types in tissue cross-sections. Can be used to classify. In another embodiment of the invention, measuring activity or expression of a protein of the invention comprises
Assessment of organ status, including organ damage following immunological or toxicological injury, using techniques known to those of skill in the art, including those described in U.S. Patent No. 6,080,551 and RE35,419.
Or it can be used to diagnose transplant rejection.

In another embodiment of the invention, the protein of the invention, or a portion thereof, or a derivative thereof, diagnoses, treats and / or treats cell proliferative disorders associated with impaired gene expression of the protein of the invention. Can be used to prevent. Such obstacles are
Benign tumor and line cancer; leukemia; melanoma; lymphoma; sarcoma; and brain, ovary,
It includes but is not limited to cancers such as bladder, colon, liver, small intestine, large intestine, breast, kidney, lung and prostate cancers. Diagnosis is by Northern blot, RT-PCR, immunoblotting, immunochemistry, enzyme linked immunosorbent assay (ELISA) as described herein.
Methods known to those of ordinary skill in the art can be used, including nucleic acids or antibodies that can detect expression of the proteins of the invention. The amount of the protein of the invention expressed in disease derived from subject samples, controls and patients, biopsied tissues, or body fluids, or cell extracts is compared to standard values. Deviation between standard and control values establishes the parameters of disease diagnosis. For prophylactic and / or therapeutic purposes, expression of the proteins of the invention can be enhanced using any of the methods known to those of skill in the art. For example, using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system (see Nelson et al., US Pat. No. 552,277), a gene such as the delivery of a protein of the present invention or a sense promoter polynucleotide construct thereof. Treatment techniques can be used. Alternatively, the proteins of the invention or fragments thereof or derivatives thereof can be administered to a subject to treat or prevent cancerous and precancerous disorders, and generally proliferative disorders. Such disorders may be dysplastic changes in tissues, dysplastic growths of ovarian, brain, colon, breast, prostate or lung tissues, dysplastic syndromes, whether or not lesions are clinically identifiable. , Polyp syndrome, colonic polyps, precancerous lesions of the cervix (ie, cervical dysplasia), esophagus, lung, dysplasia of the prostate, neoplastic prostate, breast and / or skin and related conditions (eg, , Actinic keratosis), including but not limited to syndromes represented by abnormal neoplasms. The present invention also relates to compositions and methods using the protein of the present invention or a portion thereof, or a derivative thereof to reduce drug resistance in cancer chemotherapy. Inhibition of expression and / or activity of the proteins of the invention can be achieved using any means known to those of skill in the art. In a preferred embodiment, gene therapy methods such as antisense oligonucleotides, triple helix strategies are described elsewhere in this application. In another preferred embodiment, antagonists of the protein activity of the invention can be used. These antagonists may be administered directly to the patient. Low molecular weight inhibitors (ie, those that are freely deliverable into the brain as well as specifically inhibit GST activity) are particularly preferred for use in treating cancer. Alternatively, a peptide modulator of the protein activity of the invention, or an inhibitor and a flanking sequence effective for expressing the protein coding sequence in a host cell, as well as flanking regulatory sequences (of the glutathione S-transferase in the presence of an antioxidant molecule). Artificial DNA constructs encoding (eg, antioxidant response elements that enhance expression) can be used. Such artificial DNA constructs provide the recombinant cells with increased resistance to anti-tumor agents. There is a need for selective inhibitors of GST isozymes for the treatment of drug resistance in cancer patients. Thus, in a further embodiment of the invention, the proteins of the invention or fragments thereof can be used for screening compounds which are selective or specific inhibitors of one or more GST isozymes. By selective inhibition is meant that the compound has a higher inhibitory effect on one isozyme as compared to another GST isozyme. Such compounds are also tested and selected for their ability to overcome drug resistance to chemotherapeutic agents (see Jones et al., US Pat. No. 6,103,665, August 2000). For example, mammalian cell lines that have been made resistant to a particular chemotherapeutic drug can be used to identify haloenol lactone compounds that sensitize the cell line to the chemotherapeutic drug. Such cell lines known to those of skill in the art are described, for example, in American Culture Collection, Rock.
Available from ville, Md., USA.

<SEQ ID NO: Protein GRP (187-38-0-0-l10-CS)> The sequence referred to herein as GRP, which is encoded by the DNA of SEQ ID NO: 65 referred to herein as GRP2. The protein of number 306 is homologous to bovine glutamate concentrated protein (GAPR) (GENEPEPT ID: M61185). The proteins of the invention are overexpressed in brain and fetal brain, lymph nodes and thyroid. The protein of the present invention has homology with bovine glutamate enriched protein (GARP) (
BLASTP 2.0.9 presents 18% identical amino acids, 28% positive amino acids) upon alignment. The GARP protein has been identified as a multivalent protein that interacts with those having the major roles of cGMP signaling, phosphodiesterases and guanylyl cyclases. GARP proteins are closely associated with cyclic nucleotide gated channels (CNGs) that make up a family of non-selective cation channels found in various tissues. The beta subunit of CNG has a unique bipartite structure that includes a transmembrane region (beta portion) and a GARP portion (GARP). GARP is highly homologous to GARP1, which is a soluble splice form, and splice variants lacking the C-terminal glutamate enriched region. Experiments with GARP attached to an affinity column have shown that phosphodiesterase is highly retained on the column <Korschen
HG et al., Nature, 400 (6746): 761-766 (1999)>. In addition, Korschen et al.
It was demonstrated that ARP inhibits both soluble and membrane-bound phosphodiesterase. Cyclic nucleotides are involved in controlling the activity of airway smooth muscle and numerous other cells, including proinflammatory, immunocompetent cells such as macrophages, eosinophils, mast cells and lymphocytes. Cyclic nucleotides are inactivated by the action of cyclic nucleotide phosphodiesterase enzymes (PDEs). Inhibition of cGMP PDE results in elevated cGMP levels, which are associated with beneficial antiplatelet, antineutrophil, antivasospastic and vasodilatory activities. Accordingly, the invention provides a polypeptide having the sequence of SEQ ID NO: 306, or a GRP polypeptide encoded by the human cDNA which is clone 187-38-0-0-110. In a preferred embodiment, the GRP is the sequence of SEQ ID NO: 65 or clone 187-38-.
Included are all polynucleotides that are encoded by human cDNAs that are 0-0-110 but that encode the polypeptides of the invention. As used herein, "GRP protein" includes the full-length protein of SEQ ID NO: 306, as well as bioactive fragments of the GRP protein. The “GRP protein” also includes variants of the protein of SEQ ID NO: 306 and biologically active fragments of the variant protein.

A “bioactive fragment” is defined as a peptide or polypeptide fragment of GRP that has at least one of the biological functions of a full-length protein (eg, the ability to inhibit PDE activity or be an affinity substrate for PDE). To do. The invention also provides variants of the GRP protein encoded by SEQ ID NO: 306. These variants have at least about 80% of the amino acid sequence of GRP,
More preferably at least about 90%, and most preferably at least about 95% amino acid sequence identity. The variants according to the invention also have at least one functional or structural characteristic of GRP, such as the ability to inhibit PDE activity or be an affinity substrate for PDE. The present invention also provides bioactive fragments of variant proteins. Unless otherwise stated, the methods disclosed herein can be carried out using GRP or variants thereof. Similarly, the methods of the invention can be practiced with bioactive fragments of GRP, or bioactive fragments of GRP variants. Assays for the inhibitory effects of the proteins of the present invention may be performed using any of the techniques described in US Pat. I can do it. One aspect of the present invention is in the molecular biology art nucleotide sequence of SEQ ID NO: 65,
Alternatively, compositions and methods using the complement thereof are provided. These techniques are PCR
Use of GRP2 segment as an oligomer for use; expression of GRP2 and production of recombinant protein; production of antisense RNA and DNA, their chemical analogs, etc .; Use of GRP2 segment as a hybridization probe and chromosomal gene mapping However, it is not limited to them. For example, the nucleotide sequence of SEQ ID NO: 65, or its complement, can be used to produce hybridization probes for mapping naturally occurring genomic sequences. Sequences can be mapped to specific chromosomes or specific regions of chromosomes using known techniques. These are in situ hybridization for chromosome extension, chromosome preparation by flow sorting, or artificial chromosome constructs such as yeast artificial chromosomes, bacterial artificial chromosomes, bacterial P1 constructs or Price (Price CM-Blood Rev. 1993, 7 (2)
: 127-34) and Trask B (Trask BJ-Trends Genet-1991, 7 (5): 149-54)
Includes a single-chromosome cDNA library as reviewed by.

In situ hybridization of chromosomal preparation methods and physical mapping techniques, such as linkage analysis using off-the-shelf chromosomal markers, is invaluable in extending genetic maps, which can be used for positional cloning or other genetic discovery. It provides valuable information for researchers who search for disease-causing genes using technology. The nucleotide sequences of the invention can also be used to detect differences in chromosomal location due to translocations, inversions, etc. between normal, carrier or affected individuals. Another embodiment of the invention provides a pharmaceutical composition comprising a GRP protein and a pharmaceutically acceptable carrier. These pharmaceutical compositions can be used in the prevention and / or treatment of various conditions in which inhibition of phosphodiesterases would be beneficial.
The biochemical, physiological and clinical effects of phosphodiesterase inhibitors are:
It suggests its use in various disease states where modulation of smooth muscle, kidney, hemostasis, inflammation, and / or endocrine function is desirable. Thus, the GRP protein is stable, unstable, and mutated (Prinzmetal) angina; hypertension; pulmonary hypertension; congestive heart failure; acute respiratory distress syndrome; acute and chronic renal failure; atherosclerosis; vascular Poor openness (eg, percutaneous transluminal coronary or carotid angioplasty or graft stenosis after bypass surgery); peripheral vascular disease; Raynaud's disease, platelet plasma, vascular disorders such as intermittent claudication Immune disease, multiple sclerosis; Cancer, inflammatory disease, graft-versus-host disease, Alzheimer's disease, memory deficiency, stroke, bronchitis, chronic asthma, acute lung injury, chronic obstructive pulmonary disease, allergic asthma, allergy Rhinitis; glaucoma; osteoporosis; preterm birth; benign prostatic hypertrophy; male and female erectile dysfunction; and diseases characterized by impaired gut motility (eg, hypersensitivity) It can be used in the treatment or prevention of several disorders and conditions, including but not limited to bowel syndrome. The GRP proteins of the invention may also provide beneficial antiplatelet, antineutrophil, antivasospasm, vasodilation, natriuretic, and diuretic activity when administered in a therapeutically effective amount. The GRP protein is also endothelium-derived relaxing factor (EDRF) when administered to individuals.
, Gastric NO administration, nitrovasodilators, atrial natriuretic factor (ANF), brain natriuretic agent (BNP), C-type natriuretic peptide (CNP), and endothelium-dependent relaxants such as bradykinin and acetylcholine Enhance the effect.

Another embodiment of the invention is a therapeutically effective amount of GR using suitable methods known to those of skill in the art.
Provide a method of treating male erectile dysfunction involving administration of P protein. Another embodiment of the invention provides an industrially significant method of PDE recovery comprising contacting a solution containing PDE with an immobilized GRP protein. In aspects of the invention, the GRP protein is immobilized on a solid support, allowing it to specifically bind to PDE contained in a solution or sample. The PDE is then prepared according to methods known to those skilled in the art.
It can be eluted from immobilized GRP protein (see, eg, Korschen et al., Supra). PDE
Is a commercially valuable item sold by a variety of sources. <The protein of SEQ ID NO: 302 (internal designation number 187-2-2-0-A3-CS)> The protein of SEQ ID NO: 302 encoded by the cDNA of SEQ ID NO: 61 is neurite formation and axon and dendrite growth. It is related to a neural expression protein (neuritin, Genseq accession number: W37859) which is known to be involved in. The 164 amino acid long protein of SEQ ID NO: 302 has 24 amino acids in Neuritin for the complete sequence.
% Identity. In particular, SEQ ID NO: 302 is two blocks with high homology to Neuritin (amino acids 41 to 60 of SEQ ID NO: 302 are 55 to amino acids 30 to 49 of Neuritin.
% Amino acid and 95% similarity, amino acids 66-117 of SEQ ID NO: 302 exhibit 32% identity and 57% similarity to amino acids 62-113 of Neuritin). The C-terminal portion of Neuritin (amino acids 116-142 of Neuritin) is highly hydrophobic and contains the cleavage site found in the CPI anchor protein. The protein of SEQ ID NO: 302 also has a hydrophobic C-terminus (21 of the last 30 amino acids are hydrophobic) and corresponds to the GPI anchor consensus sequence. Neuritin, also known as the plasticity-related gene number 15 (cpg-15) candidate, was separately identified from two groups derived from different cDNA libraries produced from kainate-treated hippocampal cells (Nedivi et al., Nature 363: 718-22 (1993); Naeve
Proc. Natl. Acad. Sci. USA. 94: 2648-53 (1997)). Neuritin is a secreted protein that contains a latent GPI anchor domain that is thought to anchor the protein to the target cell membrane. Neuritin is strongly expressed in the brain, especially in systems with overt developmental plasticity, including pyramidal neurons in Ammon's horn and granule cells in the hippocampal dentate gyrus. Strong expression was also observed in the layer of the retinal ganglion cells to the olfactory bulb, which is the main target of retinal ganglion cells, and also in the optic nerve layer of the superior colliculus (tectum). Observed in the cortex (Nedivi et al., Pro. Natl. Acad.
Sci. USA. 93: 2048-53 (1996); Naeve et al., Proc. Natl. Acad. Sci. USA. 94.
: 2648-53 (1997); Nedivi et al., Science). mRNA is expressed during development and persists in adulthood. Moreover, Neuritin expression is upregulated in adults by brain-derived neurotrophic factor (BDNF). Neuritin mRNA was also detected in lung and liver at lower levels than observed in the CNS ((Naeve et al., Proc. N.
atl. Acad. Sci. USA. 94: 2648-53 (1997)). Studies on the function of the Neuritin protein have revealed a role in neuronal growth. In one such study, rat cortex and hippocampal neurons were treated with a recombinant form of Neuritin. Neurons treated with Neuritin showed extensive neurite formation compared to control cultures. In particular, neurons showed well-differentiated cell bodies with well-defined spreading after neuritin treatment (Naeve et al., Proc. Natl. Acad. Sci. USA. 94:26.
48-53 (1997)). Other studies using the frog optic tectum include optic cell neuritin.
We have shown that transfection with cDNA allows to increase dendritic growth rate of optic cells (Nedivi et al., Science, 281: 1863-66 (1998)). Studies have shown that Neuritin can modify retinal optic axon growth and thus promote retinal optic synaptic maturation by increasing presynaptic axon production (
(Cantallops et al., Nature Neuroscience 3: 1004-1011 (2000)). Taken together, these results indicate that Neuritin promotes the growth of pre- and postsynaptic neurons and contributes to the formation and stabilization of mature synapses.

The invention provides a polypeptide of SEQ ID NO: 302 and a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 302. In one embodiment, the polypeptide of SEQ ID NO: 302, including fragments, variants and the like, is replaced by the corresponding polypeptide encoded by the human cDNA which is clone 187-2-2-0-A3-CS. .
The invention also includes bioactive fragments of the protein of SEQ ID NO: 302, and polynucleotide sequences encoding these bioactive fragments. In other embodiments, the bioactive fragment is at amino acid positions 41-60, 66-117, 41-117.
And 41 to 164 are included. In other embodiments, these fragments may be joined together by chemical linker segments or recombinantly inserted amino acid linkers according to methods known in the art. A "bioactive fragment" is defined as a peptide or polypeptide fragment of SEQ ID NO: 302 that has at least one of the biological functions of a full-length protein (eg, stimulates neurite formation and axon and dendrite growth). To do. The invention also provides a variant of SEQ ID NO: 302. These variants have at least about 80%, more preferably at least about 90%, even more preferably at least about 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 302. Variants according to the invention may also have SEQ ID NO: 302, such as the biological functions described above.
It has at least one function or structural characteristic of. The present invention also provides bioactive fragments of variant proteins. Unless otherwise stated, the methods disclosed herein can be performed using the polypeptide of SEQ ID NO: 302 or variants thereof. Similarly, the method of the present invention comprises a biologically active fragment of SEQ ID NO: 302 protein,
Alternatively, it can be carried out using a variant of the bioactive fragment. Due to the redundancy of the genetic code, a variety of different DNA sequences can encode SEQ ID NO: 302. It is within the skill of one in the art to create these alternative DNA sequences that encode proteins having identical or substantially identical amino acid sequences. These mutant DNA sequences are therefore within the scope of the invention. As used herein, "substantially identical sequence" means an amino acid substitution that does not substantially affect biological activity,
By a sequence with deletions, additions or insertions is meant. Fragments that retain one or more of the characteristic biological activities of SEQ ID NO: 302 are also included in this definition.

A “recombinant nucleotide variant” is an alternative polynucleotide that encodes a particular protein. They can be synthesized, for example, by taking advantage of the "redundancy" of the genetic code. Diverse codon substitutions, such as silent changes that produce specific restriction sites or codon usage-specific mutations, optimize cloning into plasmid or viral vectors, or expression in specific prokaryotic or eukaryotic host systems, respectively Can be introduced to The protein of SEQ ID NO: 302 and variants thereof can be used to raise antibodies by methods known in the art. Antibodies can be monoclonal or polyclonal. Antibodies can also be synthesized by known methods against the immunogenic fragment of SEQ ID NO: 302, as well as variants thereof. The invention also provides SEQ ID NO:
Antibodies that specifically bind to the bioactive fragment of 302 or the variant of the variant of SEQ ID NO: 302 are provided. The protein of SEQ ID NO: 302 can be used to treat central and peripheral nervous system diseases and disorders caused by modification of the protein expression pattern of SEQ ID NO: 302. In this aspect of the invention, a composition comprising the protein of SEQ ID NO: 302 and a pharmaceutical carrier is administered to an individual in need thereof. Alternatively, if the protein of SEQ ID NO: 302 is overexpressed, the reduction in SEQ ID NO: 302 levels can be achieved by various methods known to those of skill in the art. These methods include the introduction of neutralizing antibodies or the use of antisense polynucleotides derived from the protein of SEQ ID NO: 302 or clone 187-2-2-0-A3-CS. The invention also provides materials and methods for the treatment of neurological disorders, comprising contacting a neuronal cell with a composition comprising the protein of SEQ ID NO: 302 and a pharmaceutically acceptable carrier. Thus, this aspect of the invention provides a method for treating a patient suffering from various neurological disorders, conditions, and / or diseases of the central, autonomic, or peripheral nervous system. These are congenital diseases, trauma, surgery, stroke, ischemia, infectious diseases, metabolic diseases,
Includes nutritional deficiency, malignancy, and / or nerve damage caused by exposure to toxic drugs. Other examples of such disorders include tencan, cerebral neutrality,
Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neuromuscular atrophy, retinitis pigmentosa, inheritance Ataxia, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, intracranial thrombophlebitis, myelitis and spinal nerve Rootitis, viral central nervous system disease, prion disease, Creutzfeld-Jakob disease, Gerstmann-Stroisler-Scheinka syndrome, fatal familial insomnia, diabetes-induced peripheral neuropathy or other metabolic or nutritional deficiency-induced Neuropathy, neurofibromatosis, tuberous sclerosis, cerebellar retinal hemangioblastoma, cerebrospinal trigeminal syndrome, mental retardation and other central nervous system Developmental disorders, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, dermatomyositis and polymyositis, hereditary, metabolic, endocrine and toxic myopathy, severe muscle Mental disorders including asthenia, periodic paralysis, mood, anxiety, and schizophrenia, seasonal affective disorder, akathisia, amnesia, and / or other visual, sensory, olfactory, auditory, motor, or memory systems. Dystrophies or degenerative disorders of, but not limited to. Methods of introducing therapeutic compounds into cells are known to those of skill in the art. Non-limiting examples include the use of targeted liposomes, fusogenic liposomes, or other carriers suitable for the introduction of therapeutic compounds into target cells.

The present invention also provides agents and methods for the treatment of neurological disorders comprising contacting a neuron with a composition comprising a polynucleotide encoding the protein of SEQ ID NO: 302 and a pharmaceutically acceptable carrier. Supply. In one embodiment, the polynucleotide is clone 187-2-2-0-A3-CS. Methods for introducing a polynucleotide into a cell and directing expression of the polynucleotide are known to those skilled in the art. Antibodies raised against the protein of SEQ ID NO: 302 can be used in various immunoassay methods known to those of skill in the art. In this aspect of the invention, immunoassay screening for abnormal levels of the protein of SEQ ID NO: 302 can be used as a neurodegenerative disease screening or diagnostic / prognostic indicator. Antibodies raised against the protein of SEQ ID NO: 302, its fragments, and / or its derivatives were also found in pyramidal neurons of Ammon's horn and hippocampal dentate gyrus granule cells It can be used for the detection and identification of neurons, optic nerve layers of the superior colliculus (tectum), and neurons of the thalamic nucleus and cerebral cortex, but not limited to. <Protein of SEQ ID NO: 301 (187-12-4-0-A8-CS)> The protein of SEQ ID NO: 301 encoded by the cDNA of SEQ ID NO: 60 is the eukaryotic cell growth inhibitory described in International Patent Application Publication WO9617933. Factor (GENESEQP: R95950)
Is homologous to. The protein of the present invention is strongly expressed in brain, fetal brain, fetal liver and prostate. The protein of the present invention is considered to be a cell growth inhibitor. Preferred polypeptides of the present invention are those containing amino acids 221-287. Other preferred polypeptides of the invention are fragments of SEQ ID NO: 301 that have any of the biological activities described herein. In the present invention, the cytostatic factor is at least one type of cell such as, but not limited to, bacteria, yeast, vertebrate cells, mammalian cells and human cells under normal culture conditions known to those skilled in the art. Is defined as a peptide or protein that reduces, suppresses or terminates growth (reversible or irreversible). The assay for suppressive activity of the present invention can be carried out, for example, by assessing the decrease in DNA synthesis as described in WO 96/17933 or by measuring cell number or density using standard methods. For example, fibroblasts are transfected with a vector containing a DNA sequence encoding a protein of the invention or a portion thereof. The cells are then cultured in standard medium, exposed to tritiated thymidine and further cultured. The cultures were then fixed, X-Gal stained, blue stained galactosidase expressing cells were counted under the microscope and the percentage of cells showing dark particles in their nuclei due to tritiated thymidine incorporation. To decide. DN
The rate of A synthesis inhibition is defined by the reference labeling index (ie, that of a cultured cell transfected with an “empty” vector (ie, not modified to contain DNA encoding a protein of the invention or a portion thereof). 0% inhibition).

Cellular aging is associated with aging of individuals. The maximum possible number of cell divisions in culture (mitotic lifespan) is inversely proportional to individual age. When senescent cells are fused to juvenile or immortalized cells, DNA synthesis does not occur in senescent cells and, in contrast, DNA synthesis is suppressed in juvenile and immortalized cells (Stein GH et al.,-Proc Nat Acad Sci.- 1981
, 78: p3025). This demonstrates that certain factors controlling cell senescence are predominant and that senescent cells not only lack substances essential for their growth, but also have substances that actively suppress DNA synthesis. Furthermore, microinjection of mRNA prepared from senescent cells is known to suppress DNA synthesis (Lumpkin CK
,-Science-1986, 232: p393). Thus, there are several genes that are newly expressed or whose expression increases as cells age. Such genes play an important role, directly or indirectly, in cell senescence. Pereira-Smith et al. Demonstrated the existence of four groups of human senescence genes by testing the complementation of large numbers of fusion pairs in immobilized human cells (Pereira-Smith OM et al., Proc N.
at Acad Sci.-1988, 85: p6042). Clarifying the essence of senescence-related genes not only understands senescence at the cellular and individual levels, but the use of these genes or gene products also contributes to the diagnosis of various aging-related diseases and diseases caused by cellular senescence, It is of interest to enable the development of prophylactic / therapeutic agents for such diseases, and the prophylactic / therapeutic agents for a variety of diseases, including but not limited to cancer, including uncontrolled cell growth. In one embodiment of the invention, the polypeptide or polynucleotide of the invention specifically labels the tissue cells of brain, fetal brain, fetal liver and prostate cell tissues, since the protein is strongly expressed. Used to The ability to specifically detect these tissues, and cells derived from these tissues, has several applications, including determining tumor cell history and tissue analysis. Embodiments of the present invention relate to methods and compositions that use the protein of SEQ ID NO: 301 or the cDNA of SEQ ID NO: 60 or a portion thereof to suppress in vitro cell proliferation. For example, the present invention is a "cocktail" with other proteins (proteases, etc.)
By including it, it can be used as a decontaminating agent that prevents the growth of cells to maintain a sterile environment. A preferred application of this embodiment is when the present invention is used as a bacteriostatic / fungal growth inhibitor, a sample (such as a cell culture medium) and an instrument (
Decontamination of surgical instruments). Other examples include, for example, as reagents for synchronization of cell division, terminating the cell cycle of cultured cells at a predetermined point in time and using techniques such as flow cytometry to identify specific cells (eg, desired cells in culture) in culture. Of the protein of the invention as a reagent for avoiding the need to isolate Cell cycle synchronization can be achieved, for example, by transfecting cells with a suitable vector containing DNA encoding a protein of the invention, or a portion thereof, such that expression of the protein results in growth inhibition. Then, after a period of time, allowing cells to resume growth (eg, simultaneously in the S phase where DNA is synthesized),
An inhibitor of the protein can be administered. In addition, the ability to synchronize the cell cycle in in vivo experimental systems provides advances in assay accuracy and facilitates research methods or experiments related to particular cell cycle stages. The use of the present invention for the in vitro inhibition of cell proliferation is not limited to the above examples, and the present invention may be useful in any in vitro application that requires inhibition of cell proliferation.

Other embodiments of the present invention include SEQ ID NO: 301, or SEQ ID NO: 60, or a portion thereof into a target tissue (such as skin or vascular endothelium) to establish an in vitro senescent cell line of the target tissue. Regarding the introduction of. Such cell lines provide an in vitro model of senescent cells (in vitro cell culture offers the advantage of being easy to produce and not expensive as in animal models), of senescence and / or of cellular senescence. The mechanism is clarified, and it is useful as a screening system for the prevention and treatment of diseases related to aging and / or diseases caused by cellular aging. Thus, these cells may be useful in drug candidate screening applications, with preferred applications including use in high throughput screening, for example to identify "lead compounds". A preferred embodiment of the present invention relates to the use of the cDNA of SEQ ID NO: 60 or a part thereof as a probe for testing aging of individuals at the gene expression level. In particular, SEQ ID NO: 60 or part thereof is used as a diagnostic reagent for various aging-related diseases such as but not limited to arteriosclerosis, osteoarthritis, dementia (including Alzheimer's disease) and Parkinson's disease. it can. In a related embodiment, the cDNA of SEQ ID NO: 60, or a portion thereof, can be used to synthesize antisense oligonucleotides by methods known to those of skill in the art.
Antisense oligonucleotides can be used to inhibit the synthesis of the protein of SEQ ID NO: 301, thereby preventing the aging of cells and tissues and / or promoting the rejuvenation of aging cells and tissues. Antisense oligonucleotides may also be used in vivo or ex vivo in aging or diseases caused by aging-related diseases such as, but not limited to, arteriosclerosis, osteoarthritis, dementia (including Alzheimer's disease) and Parkinson's disease. It can be used for treatment and prevention. In the most preferred embodiment, SEQ ID NO: 301, SEQ ID NO: 60, or a portion thereof,
It can be used as a medicament for the treatment of conditions such as, but not limited to, cancer, inflammation or infection. For example, when used as an antibacterial, antiviral and / or antifungal agent, the inhibition of microbial growth directly inhibits the growth of microorganisms (in the case of fungal and bacterial infections) or viral infections. The case is achieved by suppressing DNA synthesis in infected cells. The DNA of SEQ ID NO: 60, or a portion thereof, can also be used to develop gene therapy products in the in vivo or ex vivo treatment of diseases and conditions such as cancer and inflammation. SEQ ID NO: 301, or SEQ ID NO: 60, or a portion thereof, may also be used in 1] diagnostic probes, 2] prophylaxis, or 3] arteriosclerosis, osteoarthritis, dementia (including Alzheimer's disease) and Parkinson's disease, etc. However, it can be used as a treatment for, but not limited to, aging-related diseases.
In a related embodiment, the protein of SEQ ID NO: 301 or the cDNA of SEQ ID NO: 60, or a portion thereof, may be associated with diseases described above (eg, diseases caused by cell senescence, diseases related to senescence, and diseases caused by cell proliferation). ) Can be used for the development of a drug that can be used for prevention or treatment.

For treating or preventing diseases and conditions associated with unwanted proliferation such as cancer, inflammation, or infection, several methods can be used to increase the expression or activity of the protein. For example, a polynucleotide encoding a protein can be introduced into an unwanted cell where it expresses the protein and suppresses further cell growth. In one such embodiment, the polynucleotides are incorporated into liposomes that include on their surface specific molecules (eg, tumor-specific antibodies) that direct the liposomes to target specific cell types. Alternatively, the protein of SEQ ID NO: 301 itself can be administered to cells, eg, as a fusion protein that also includes a specific targeting polypeptide moiety. In addition, a compound that enhances protein expression or activity is preferably administered to cells, such as chemically linked to a heterospecific targeting molecule, to specifically target said compound to unwanted cells. Good. <Protein of SEQ ID NO: 412 (internal designation number 187-5-3-0-C7-CS)> The protein of SEQ ID NO: 412 encoded by the cDNA of SEQ ID NO: 171 is human CD
It is homologous to the K4-binding protein p34 SEI-1 (sptrembl accession number Q9UHV2). p34S
EI-1 is a new CD that prevents the inhibition of cyclin D1-CDK4 complex formation by p16INK4a
It is a K4 regulator. p34SEI- appears to act as a growth factor sensor to promote the formation and activation of the cyclin D-CDK complex despite the inhibitory level of INK4 protein (Sugimoto et al., Genes Dev. 13: 3027-3033). (1999)). Cell cycle progression is a complex process regulated at multiple levels by several proteins. The activity of cyclin-dependent kinases (CDK4 and CDK6) is driven by cyclin partners, which act as positive effectors, and two families of cdk inhibitory proteins (KIPs), and p16I, which acts as negative effectors.
It is regulated by the association of inhibitors of cdk4 (INK4) like NK4a. Cancer is a disease characterized by loss of cell growth control, and the molecular mechanisms of the cell cycle are involved in tumorigenesis. Many human tumors show abnormalities in this pathway, resulting in functional inactivation of p16INK4a or CDK4 overactivity (Palmer
o at al., Cancer Surv. 27: 351-357 (1996)). The protein of SEQ ID NO: 412 binds to a cyclin-dependent kinase,
It is believed to be involved in cell cycle control. Other preferred polypeptides of the present invention are fragments of SEQ ID NO: 412 having any of the biological activities described herein. The binding activity of the proteins of the invention, or portions thereof, to cyclin-dependent kinases, as well as their role in the cell cycle, may be determined using any of the assays known to those of skill in the art, including those described by Sugimoto et al. (Supra). Can be tested.

Embodiments of the present invention relate to cyclin dependent kinases, preferably CDs, in biological samples.
Methods of using the proteins of the invention or portions thereof to identify and / or quantify K4, and therefore use in assay and diagnostic kits for the quantification of such CDKs in body fluids, tissue samples, and mammalian cell cultures. To be done. The binding activity of the proteins of the invention or portions thereof can be assessed using the assays described by Sugimoto et al. (Supra) or other methods known to those of skill in the art. Preferably, under a condition capable of forming a complex between the protein of the present invention or a part thereof and a cyclin-dependent kinase to be identified and / or quantified, a defined amount of the protein of the present invention or one thereof is used. Parts are added to the sample. The presence of the complex and / or free protein of the invention or part thereof is then assayed and compared to controls using methods known to those of skill in the art. In another embodiment, the invention relates to compositions and methods that use the proteins of the invention or portions thereof to stimulate cell proliferation in vitro and in vivo. For example, an effective amount of a protein of the invention or a soluble form of a portion thereof to stimulate cell proliferation may be added to the cell culture medium. The present invention further includes line cancer, sarcoma, lymphoma, leukemia, melanoma, myeloma, teratocarcinoma, adrenal gland, bladder, bone, brain, breast, digestive tract, heart, kidney, liver, lung, ovary, pancreas, parasite. Of several disorders associated with cell proliferation, including but not limited to neurodegenerative disorders such as ganglion, parathyroid gland, prostate, salivary gland, skin, spleen, testis, thyroid, uterine cancer, and Alzheimer's disease. Methods and compositions using the proteins of the invention or parts thereof for diagnosis, prevention and / or treatment (
McShea et al., Am. J. Pathol. 150 (6): 1933-1939 (1997)). For diagnostic purposes, quantification of the proteins of the invention can be investigated in biological samples using any of Northern blotting, RT-PCR, immunoblotting and protocols known in the art,
Compared to expression in a control biological sample. Therefore, diagnostic assays can be used to determine the modified expression of the proteins of the invention to assess disease association and prognostic significance of disease. For prophylactic and / or therapeutic purposes, using either the antisense or triple helix methods described herein,
Inhibition of endogenous expression of the proteins of the invention can be used. Alternatively, inhibitors of protein activity can be developed and used to inhibit and / or reduce its activity using any of the methods known to those of skill in the art. Antibodies that specifically bind the proteins of the invention can be produced using methods known to those of skill in the art and used as antagonists.

<Protein of SEQ ID NO: 299 (Internal Designation No. 184-1-4-0-C11-CS)> The protein of SEQ ID NO: 299 encoded by the cDNA of SEQ ID NO: 58 was found in fetal liver and liver, BolA protein is orthologous. The BolA family includes the morph protein BolA from E. coli and its diverse homologues.
BolA expression is growth rate regulated and is induced during the transition to stationary phase. BolA
Is also induced by early growth stress and has a general role in the stress response. It has also been suggested that BolA is able to induce transcription of penicillin binding proteins 6 and 5 (EMBO J. 1; 8 (2): 3923-31 (1989)). E. coli cells become thin and short morphology after post-starvation or stationary phase conditions, and this morphological change is an adaptive response of E. coli to common stress types. The bolA gene appears to be involved in the switch between cell elongation and the septal formation system during the cell division cycle <J Bacteriol. 170: 5169-5176 (1988)>. Regulation of bolA has been linked to the presence of a gearbox promoter in which RNA is transcribed <Mol Microb
iol. 5: 2085-2091 (1991)>. Expression of bolA is driven by two promoters. P2 is located further upstream from the structural gene, under the control of σd, and transposes bolA constitutively. Promoter P1 proximal to the structural gene is a σ s -controlled gearbox promoter in which bolA has been shown to be transcribed in a reverse growth rate-dependent manner <
J Bacteriol. 173: 4474-4481 (1991)>. The alternative sigma factor σ s, encoded by the gene rpoS, has been described as a central regulator of a series of specific gene inductions involved in adaptation to stationary phase. Nevertheless, the σs function is not restricted to the stationary phase. A significant increase in cellular level σ s was observed during exponential growth in response to stress, and the genes under its control code for important adaptive regulators of general stress conditions <FEMS Micro
biol Lett 30: 419-430 (1997)>. The small form resulting from stress-induced overexpression of bolA reduces the surface area exposed to the environment and reduces the surface-to-volume ratio of cells. The identification of orthologous genes provides important information about function and structural conservation within these orthologs throughout evolution. The concept of comparative gene identification has been used by numerous laboratories to search for orthologous genes once the particular gene of interest has been identified in another species <Genome Res. 10 (5): 703- 13 (2000
)>.

The protein of the invention contains a signal peptide corresponding to the short helix as predicted by the software TopPred II <Clarosand and von Heijne, CAB.
IOS applic. Notes, 10: 685-686 (1994)>. Accordingly, one aspect of the invention provides materials and methods for delivery of recombinant proteins to hepatocytes. The signal peptide encoded by the appropriate polynucleotide may bind to other proteins / polypeptides (also encoded by the appropriate polynucleotide). A recombinant gene containing a signal peptide sequence is expressed to deliver the desired protein via the signal peptide. Methods of producing such gene fusions, expression of such gene products, and their uses are known to those of skill in the art. In other embodiments, the BolA or bioactive fragment thereof is a cytotoxic agent,
It can be used to modulate the stress response to environmental changes such as heat shock, irradiation, genotoxic stress or growth factors. In another aspect of the invention, SEQ ID NO: 299 is incorporated into a prokaryotic expression vector and transfected into prokaryotic cells that are unable to adapt to environmental stress, or cells containing a BolA deficiency. The expression vector optionally comprises a promoter system (eg, P1, P2, σd, etc., as described above) that generally controls bolA expression.
σs etc.) can be included. The components required for transcription are provided in one or more expression vectors. Prokaryotic cells transformed in this way may be useful in bioremediation systems, which normally encounter environmental stress. Accordingly, preferred prokaryotes for practicing this aspect of the invention are known to be bolA deficient or deficient in bolA and useful for bioremediation. In other embodiments, the invention provides methods and compositions for selectively identifying liver tissue. The protein encoded by SEQ ID NO: 299 can be used for specific polyclonal or monoclonal antibody synthesis using techniques known to those of skill in the art. These antibodies can be used to selectively identify liver tissue by known histological immunoassays. The ability to immunologically identify a tissue sample can be used to analyze a mislabeled biopsy sample (eg, laboratory error) questioning the origin of the tissue sample, or simply to confirm that the tissue sample originates from the liver. Industrially important. Antibodies can also be used to identify liver primary cancer metastases.

In addition, the antibodies provided by the present invention can also be used to assay animal feed for the presence of liver or liver byproducts. As is known, many animal feeds contain animal proteins. The use of animal feeds containing animal proteins is associated with disorders in both animals and humans (most notably bovine spongiform encephalitis). This resulted in a ban on animal protein in animal feed. However, animal feed must be tested to ensure such a ban. Thus, the antibodies of the invention can be used to test animal feed for the presence of liver by methods known to those of skill in the art. <Proteins of SEQ ID NOs: 249 and 288 (internal designation numbers 105-037-2-0-H11-CS and 174-7-4-0-H1-CS)> SEQ ID NO: 249 encoded by the cDNA of SEQ ID NO: 8 The 403 amino acid long protein is SEQ ID NO: 4 with the exception of the 5 amino acids at positions 192-194 and the absence of the amino acids at positions 298-299 in the protein of SEQ ID NO: 288.
It has extensive homology with the protein of SEQ ID NO: 288 encoded by the cDNA of 7. The two proteins appear to be the result of alternative splicing,
Present similar function and utility. The 403 amino acid long protein of SEQ ID NO: 249 overexpressed in salivary glands exhibits extensive homology with the mouse muscle hypothetical protein (Genbank accession number AB030196).
Amino acid residues in the protein of SEQ ID NO: 249 show a high degree of identity with the Genbank sequence. However, the protein of the Genbank sequence does not have 20 amino acids (192-194, and 298-303, 353-354, 380-381 and 387-393), and also 35 amino acids that differ from SEQ ID NO: 249. Presents amino acids. Furthermore, the protein of SEQ ID NO: 249 contains 31-51, 75-95, 154 predicted by the software TopPred II.
Four transmembrane domains from positions ~ 174 and 236-256 are predicted II (
Claros and von Heijne, CABIOS applic. Notes, 10: 685-686 (1994)). When expressed in E. coli, the corresponding sequences suppress bacterial growth (Inoue et al., Bioche
Biophys. Res. Commun. 268: 553-61 (2000)). Therefore, SEQ ID NO: 249 and
It is believed that the 288 protein or bacterial growth inhibiting fragment thereof can be used to inhibit bacterial growth by contacting the polypeptide of the invention with bacteria (gram negative or gram positive). The growth inhibitory activity of the proteins of the invention, or portions thereof, can be assayed using any of the assays known to those of skill in the art, including those described in Inoue et al. (Supra).

According to one aspect of the invention, methods and compositions are provided that use the proteins of the invention or fragments thereof to inhibit bacterial growth. In a preferred embodiment, the proteins of the invention are expressed in bacteria, preferably E. coli, using recombinant DNA technology methods known to those skilled in the art. The expressed protein is then used to suppress bacterial growth. The effects of the expressed protein and its analogs or antagonists can be assessed using methods or techniques known to those of skill in the art. Also included in the invention is the polypeptide encoded by the human cDNA which is clone 105-037-2-0-H11-CS-SD. The polypeptide of SEQ ID NO: 249 may be compatible with the polypeptide encoded by the human cDNA corresponding clone 105-037-2-0-H11-CS-SD. Polynucleotides encoding the above polypeptides are also included in the invention. A preferred polynucleotide is that of SEQ ID NO: 8 and the human cDNA of clone 105-037-2-0-H11-CS-SD. Nucleotide sequences encoding the polypeptides of SEQ ID NOs: 249 and 288 are used for probe generation for detection of related genes. Vectors expressing the nucleotide sequences can be used to express the polypeptide in target cells. Antisense nucleotides can be used to suppress the expression of polypeptides. Thus, in another embodiment of the invention, the protein or fragment thereof is
It can be used as a marker protein to selectively identify tissues, preferably salivary glands. For example, the proteins of the invention or fragments thereof can be used to synthesize specific antibodies using techniques known to those of skill in the art. Such tissue-specific antibodies can then identify tissue of unknown origin, such as differentiated tumor tissue that has metastasized to forensic samples, different body sites, etc., or use immunochemistry to identify different tissue types in tissue cross-sections. Can be used to classify. In another embodiment, the polynucleotide encoding the protein of SEQ ID NO: 249 can be used for in situ hybridization. Transcripts encoding the protein gng3lg (Genebank accession number AF069954) are divergent across the novel human G-binding protein gamma-3 (HGPG) (Genbank accession number AF0
69953), transcribed from a bidirectional promoter with a transcript encoding a gamma3 subunit protein called gng3, a mechanism conserved across species within the human genome (Downes et al., Genomics, 53). : 220-230 (1998)).

Several genes associated with common physiological functions are αB crystallin and α
Share common divergent bidirectional promoters such as glycalin, collagen type IV A1 and A2, surf1-3 and -surf5, dihydrofolate reductase and 2 mismatch repair 1 ((Iwaki et al., Genomics, 45 386- 394 (1997), Burbelo et al.
Proc. Natl. Acad. Sci. USA 85: 9679-9682 (1988), Kaytes et al., J. Biol. Chem.
, 263: 19274-19277 (1988), Poschl et al., EMBO J., 7: 2687-2695 (1988), Soinin.
en et al., J. Biol. Chem, 263: 17217-17220 (1988), Garson et al., Genomics, 30:16.
3-170 (1995), Williams et al., Mol. Cell. Biol., 6: 4558-4569 (1986), Fujii.
J. Biol. Chem., 264: 10057-10064 (1989)). A family of GTP-hydrolyzing enzymes, heterodimeric G-proteins, are present in all cells and by transducing hormones, neurotransmitters and sensory signals into an array of cellular responses,
Controls various functions (metabolic, humoral, nervous and developmental). Triggered by cell surface receptors, each G protein produces an adenylate cyclase, phospholipase C,
And regulates the activity of specific effectors, including ion channel proteins that elicit appropriate biochemical responses. In view of this, the transcript encoding the protein of SEQ ID NO: 249 shares common regulatory elements with the gng3 gene and is
Such gene products, which are proteins of gng3 and gng3, are thought to be coupled in a physiologically unknown manner. Therefore, in an embodiment of the present invention, SEQ ID NO: 249
Proteins or parts thereof are used to control the transmission of hormones, neurotransmitters and sensory signals to provide an array of cellular responses. In other embodiments of the present invention, the polypeptides of the present invention and related polynucleotides include, but are not limited to, cancer, neurodegenerative diseases, cardiovascular disorders, hypertension, renal injury and repair, septic shock. , Can be used to treat several types of disorders. In one embodiment, the protein of SEQ ID NO: 249 or fragments, derivatives or analogs thereof can be administered to a subject for the treatment or prevention of disorders associated with decreased expression or activity of the protein. In a further embodiment, a vector capable of expressing the protein of SEQ ID NO: 249 is
It may be administered to a subject for the treatment or prevention of disorders associated with reduced expression or activity of the protein. Naked nucleotides encoding the protein of SEQ ID NO: 249 can also be used.

[0229] In yet another embodiment, a pharmaceutical composition comprising a substantially purified protein of SEQ ID NO: 249 is administered to a subject for treatment or prevention of disorders associated with reduced expression of the protein. Good. In yet another embodiment, for antagonist development and screens,
The polypeptide of SEQ ID NO: 249 may be used. For example, a purified polypeptide is
It can be used to screen a drug library to identify those that generate antibodies or suppress the physiological function of the protein. Thus, in a further embodiment, an antagonist of the protein of SEQ ID NO: 249 may be administered to the subject for the prevention or treatment of disorders associated with increased expression or activity of the protein. Similarly, in other embodiments, a vector expressing the complement of the polynucleotide sequence encoding the protein of SEQ ID NO: 249 may be administered to reduce expression of the protein. The protein of SEQ ID NO: 249 exhibits a leucine zipper located near its NH2-terminal part (positions 20-41). It is believed that the protein of SEQ ID NO: 249 may dimerize by itself (homodimerization) or a heterologous protein of interest (heterodimerization) and its leucine zipper domain. Preferred polypeptides of the present invention are polypeptides that include leucine zipper domain fragments and fragments that have any of the biological activities described herein. The multimerizing activity of the proteins of the invention, or portions thereof, is assayed using any of the assays known to those of skill in the art, including circular dichroism spectroscopy and thermal melting analysis, as described in US Pat. it can. The utility of proteins containing a leucine zipper domain, such as the protein of SEQ ID NO: 249 is described elsewhere in this application. <Protein of SEQ ID NO: 259 (internal designation number 114-016-1-0-H8-CS)> The protein of SEQ ID NO: 259 referred to as HOPP in the present specification is clone 114-0.
It is encoded by 16-1-0-H8-CS (SEQ ID NO: 18). This protein is homologous to Arabidopsis (ASY1) and yeast (HOP1) proteins (Caryl AP et al., C
hromosoma, 109, 62-71; Hollingsworth NM et al., Cell, 61, 73-84). In addition, the 394 amino acid long protein of SEQ ID NO: 259 is pfam from position 22-230.
Present the HORMA domain. This HORMA domain is a mitotic checkpoint,
It is a common structural element in chromosome pairing and DNA repair. For example, HORMA
The domain is (1) a major component of the spindle assembly checkpoint, MAD2 (Straiht AF. Current Biology 1997, reviewed by 7: 613-616); (2
) HO, a conserved protein involved in meiotic synaptonemal complex assembly
P1 (Hollingsworth NM et al., Cell, 61, 73-84); and (3) and (3) translations,
Rev7p (Aravind L. and Koonin EV, Trends Biochem Sci. 1998 Au, a subunit of the yeast DNA polymerase “epsilon” involved in template-independent DNA synthesis
g; 23 (8): 284-6).

[0230] Meiotic homologous chromosome pairing is the synaptonemal complex (SC), a ribbon-like, protein-like structure that holds homologous chromosomes in close juxtaposition over their entire length.
Is reached in the formation of. The synaptonemal complex (SC) is a prominent, evolutionarily highly conserved structure found only in meiosis. Evidence from the mutant phenotype supports the hypothesis that recombination and SC formation are interdependent processes. First, although not required for homology recognition, SCs facilitate interactions between homologues in situations where normal processes are defective (eg interlocking, heteropairing, etc.). Second, multimerization of SC components allows the recombination process to proceed by modulating the number of pair exchanges and localization with each other (ie, interference). Third, SCs may play important roles in meiotic chromosomal structure, and especially in sister-to-sister interactions. Homologous chromosome pairing is a major event in meiosis because it is essential for normal chromosome segregation and is involved in controlling crossover frequency. (For an overview,
See Zickler D., J Soc Biol 1999; 193 (1): 17-22). Mutants in HOP1 and ASY1 are both proteins with significant homology to the protein of SEQ ID NO: 259 and exhibit reduced meiotic cross-levels and intragenic recombination between markers on homologous chromosomes (Hollingsworth NM, Byers B., Geneti
cs 1989 Mar; 121 (3): 445-62; Caryl AP et al., Chromosoma 2000; 109 (1-2): 62.
-71). Accordingly, the present invention provides HOPP polypeptides or clone 114-016-1-0-H8-CS.
A composition comprising a polynucleotide encoding a HOPP polypeptide encoded by, or by administration of a therapeutically effective amount of the polynucleotide to restore normal chromosome segregation in cells, clone 114-016-1-0-H8- Methods and compositions using the protein encoded by CS or the polynucleotide of SEQ ID NO: 18, or bioactive fragments thereof. Loss of normal chromosome segregation in normal cells leads to aberrant chromosome segregation events that are characteristic of tumor progression. The HOPP protein encoded by clone 114-016-1-0-H8-CS (SEQ ID NO: 18) is
It can be targeted to the nucleus by a nuclear targeting sequence according to known methods. Nuclear target sequence (
Alternatively NLS) can be attached to HOPP chemically or recombinantly. Or HOPP
The gene may be used in known gene therapy protocols for restoration of normal chromosomal function.

Infertility due to gametogenesis is often associated with autosomal structural abnormalities. Recent studies on the meiotic phase of meiosis have shown defects near breakpoints, association of translocation morphologies by sex chromosomes, and frequent involvement of acrocentric chromosomes. Two major models have been proposed to explain the male sterile effect of rearrangement. Spermatogenesis dysfunction can be the result of 1) XY autosomal interactions; or 2) disruption near breakpoints during the thick thread stage. These defects may be significantly involved in germ cell atresia (for review, see Luciani JM, Guichaoua MR Reprod Nutr De
v. 1990; Suppl 1: 95s-103s and Miklos GL. Cytogenet Cell Genet. 1974; 1
3 (6): 558-77)). Accordingly, the present invention also provides SEQ ID NO: HOPP or clone 114-016-1-0-H8-C to reduce the incidence of infertility due to gametogenesis.
Methods and compositions using proteins of S, or biologically active fragments thereof. HOPP can be introduced into sperm as described in the previous paragraph. NLS
HOPPs that are optimally linked to sequences can also be introduced into sperm or eggs by other methods known in the art, such as electroporation or microinjection. The protein of SEQ ID NO: 259, encoded by clone 114-016-1-0-H8-CS, may also arrest cell division in human cells if the spindle is improperly attached to the chromosome (Allshire RC Current Opinion in Genetics and Development 1997
, 7: 264-273). In the absence of the functional protein of SEQ ID NO: 259, cells exposed to drugs that inhibit spindle formation, such as benomyl, vinblastine, and nocodozole, are predicted to undergo rapid cell death due to massive chromosome loss. It Human cells containing HOPP are expected to survive despite such drug treatment because they are able to arrest division prior to a chromosome loss event. Tumor cells that are hypersensitive to chemotherapeutic drugs that inhibit spindle formation may be sensitive to these drugs because they lack checkpoint proteins. Therefore, screening assays for the presence or absence of this protein in a given tumor provide an indication of chemosensitivity of the particular tumor. The present invention thus provides a prognostic effect of treating a patient with a chemotherapeutic drug,
Or a method for determining which of the chemotherapeutic agents from at least two groups is more effective for the patient. Furthermore, loss of checkpoint function in normal cells can predispose them to aberrant chromosomal segregation events that are characteristic of tumor progression. Thus, the antibodies, polypeptides and polynucleotides of the invention are useful in diagnosing certain cancers.

Polyclonal antibodies can be produced by injecting rabbits, rats, goats, mice or other animals with the immunogens of the invention. Serum is extracted from the host animal and screened to obtain polyclonal antibodies specific for the immunogen.
Polyclonal antibody screening methods are known to those of skill in the art and can be found in Harlow & Lan.
e, Antibodies: Experiment Manual (Antibodies: A Laboratory Manual) (Cold Spring
Harbor Laboratories, Cold Spring Harbor, NY: 1988), the contents of which are incorporated herein by reference. Monoclonal antibodies can be produced, for example, by immunizing mice with the immunogen according to the invention. Monoclonal antibody production methods are known in the art and are
hler, B. and Milstein, C., Nature (1975) 256: 495-497.
The hybridomas can, if desired, be expanded and the supernatant assayed by conventional immunoassays, eg, radioimmunoassays. Positive clones can be further characterized. Hybridomas producing the desired antibody can be grown in vitro or in vivo using known methods. Monoclonal antibodies can be isolated by conventional immunoglobulin purification methods such as ammonium sulfate precipitation, gel electrophoresis, dialysis, affinity chromatography, ultrafiltration and the like. The antibody of the present invention can be labeled with a detectable moiety. "Detectable part" as described above
Are known to those of skill in the art and include, but are not limited to, fluorescent labels, radioactive atoms, paramagnetic ions, biotin, chemiluminescent labels, or labels that are detectable via secondary enzymes or conjugation steps. Not done. The invention further provides a method for determining the sensitivity of a tumor sample to treatment with a spindle inhibitor, said method comprising: (a) labeling the tumor sample with a detectable moiety, further comprising Contacting with an antibody capable of specifically binding to the fragment and (b) assaying for the presence of the immune complex formed in step (a). The absence of immune complexes indicates that the tumor is susceptible to treatment with spindle inhibitors such as benomyl, vinblastine, nocodozole. The invention also provides a pharmaceutical composition comprising a nucleic acid encoding the protein of SEQ ID NO: 259 and a carrier. In one aspect of the invention, the composition is capable of crossing cell membranes and provides for expression of the proteins of the invention. As used herein, "carrier" includes pharmaceutically acceptable carriers, including phosphate buffered saline, water, emulsions such as oil / water emulsions or triglyceride emulsions, various types of wetting agents, tablets. , Coated tablets and capsules, starch, milk, sugar, certain clays, gelatin, steensic acid, talc, vegetable fats or oils, gums, glycols or other known excipients such as excipients It includes any of the standard pharmaceutically acceptable carriers such as carriers. Flavoring and coloring additives or other ingredients may also be included. In addition to the standard features of pharmaceutically acceptable carriers, "suitable" carriers of the present invention may also include carriers that are capable of penetrating the cell membrane. Thus, in one embodiment of the pharmaceutical composition, the pharmaceutically acceptable carrier, after binding to its structure, binds to a receptor on the cell that can be taken up by the cell.

The invention further provides a method of inhibiting tumorigenesis in a subject, comprising administering an effective amount of a nucleic acid encoding a protein of the invention to enhance expression of the protein. <Proteins of SEQ ID NOs: 311 and 312 (internal designation number: 188-28-4-0-B12-CS.corr
And 188-28-4-0-B12-CS.fr)> clone 188-28-4-0-B12-CS human cDNA or the cDNA nucleotide sequence of SEQ ID NO: 70, 466 of SEQ ID NO: 311 Amino acid long proteins are no
It is associated with the proliferating cell nucleolar antigen p120 encoded by 11 (Genebank accession number M32110) and the yeast nucleolar protein Nop2P encoded by nop2 (Genebank accession number U12141). SEQ ID NO: 311 (encoded by clone 188-28-4-0-B12-CS.corr) is the three proteins (Genban described as homologues of p120.
High homology with Accession No. AK002229 and Geneseqp Accession No. Y86441, Y86442). Further, the protein of SEQ ID NO: 311 is a polymorphic variant of SEQ ID NO: 312 encoded by the cDNA of SEQ ID NO: 71. Furthermore, the proteins of the present invention display the pfam NOL1 / NOP2 / sun family signature from positions 201-276. This motif is also found by emotif from positions 230-245. The NOL1 / NOP2 / sun family includes p120 and Nop2p.
These proteins are involved in nucleolar structure and activity, as well as cell cycle regulation. Freeman JW et al. (Cancer Res. 48: 1244-51, 1988) identified p120, a 120-kD nucleolar antigen associated with proliferating cells. This protein is a growth-associated antigen that is transiently regulated during the cell cycle and demonstrates a marked increase in expression at the G1-S boundary. This suggests that p120 may be involved in cell cycle regulation and increased nucleolar activity associated with cell proliferation (Fonagy A. et al. (1993) J. Cell. Physiol.
154: 16-27). Human p120 protein is also the most cancer-specific of the growth-associated nucleolar proteins identified. Antigen p120 was detectable in a wide range of human malignancies but not benign tumors or corresponding normal tissues. The antigen was not detected in growth arrested cells and was expressed early in G1 of the cell cycle.

Overexpression of human p120 leads to transformation of NIH3T3 cells. Antisense p120
Expression of the construct restores p120 transformed cells to their original phenotype. Perlaky
L. et al <Cancer Res. 52: 428-36 (1992)> and Valdez et al <Cancer Res. 52.
: 5681-87 (1992)> reported that the intermediate region of antisense p120 suppressed proliferation of NIH 3T3 cells to about the same extent as the full-length antisense construct. The predicted mouse and human P120 proteins are 63% identical. Nop2p, another protein in the Nol1 / Nop2 / Sun family and encoded by the gene NOP2, is involved in nucleoli during initiation of growth and maintenance of nucleolar structure (de Beus et al., (1994) J Cell Biol. 127: 1799-813). Two proteins, p12
0 and Nop2p can associate with ribosomal mRNA in preribosomal particles to mediate the ribosome maturation process (Hong B. et al. (1997) Mol. Cell Biol. 17).
: 378-88; Gustafson WC et al., (1998) Biochem. J. 331: 387-93). The present invention provides a polypeptide encoded by the human cDNA clone 188-28-4-0-B12-CS and a polynucleotide sequence encoding the same amino acid sequence. The invention also includes bioactive fragments of the protein encoded by the human cDNA which is clone 188-28-4-0-B12-CS and the polynucleotide sequences encoding these bioactive fragments. "Bioactive fragment" means
It is defined as a peptide or polypeptide fragment that has at least one of the biological functions of a full-length protein (eg, the ability to transform a cell line in vitro). The present invention is also SEQ ID NO: 3, encoded by clone 188-28-4-0-B12-CS.
Provides 11 protein variants. These variants are clones 188-28-4-0-B
At least about 80%, preferably at least about 90%, and most preferably at least about 95% amino acid sequence identity to the amino acids encoded by 12-CS. The variant according to the invention also has at least one functional or structural characteristic of the protein encoded by the clone clone 188-28-4-0-B12-CS.
The present invention also provides bioactive fragments of variant proteins. Unless otherwise indicated, the methods disclosed herein include clone 188-28-4-0-B12-CS, or the protein encoded by clone 188-28-4-0-B12-CS, or variants thereof. Can be done using. Similarly, the method of the present invention uses clone 188-28-4-0-B12-C.
S, or a bioactive fragment of the protein encoded by clone 188-28-4-0-B12-CS, or a variant of said bioactive fragment.

Due to the redundancy of the genetic code, a variety of different DNA sequences have been cloned into clone 188-28-4-0-.
It can encode the amino acid sequence supplied by B12-CS. It is within the skill of one in the art to create these alternative DNA sequences that encode proteins having identical or substantially identical amino acid sequences. These mutant DNA sequences are therefore within the scope of the invention. As used herein, a "substantially identical" sequence means a sequence having amino acid substitutions, deletions, additions or insertions that does not substantially affect biological activity. Also included in this definition are fragments that retain one or more characteristic biological activities of the protein encoded by clone 188-28-4-0-B12-CS. A "recombinant nucleotide variant" is an alternative polynucleotide that encodes a particular protein. They can be synthesized, for example, by taking advantage of the "redundancy" of the genetic code. Diverse codon substitutions, such as silent changes that produce specific restriction sites or codon usage-specific mutations, optimize cloning into plasmid or viral vectors, or expression in specific prokaryotic or eukaryotic host systems, respectively Can be introduced to In one aspect of the invention, SEQ ID NO: 311 and variants thereof encoded by clone 188-28-4-0-B12-CS may be used for polyclonal or monoclonal antibody production. Fragments with both the amino acid sequence or the biological activity and immunogenicity of the variant protein can be used for antibody production. Polyclonal and / or monoclonal antibodies can be produced by methods known to those of skill in the art. The antibodies produced in accordance with the present invention can be used in various detection assays known to those of skill in the art. Another aspect of the invention provides monoclonal and polyclonal antibodies that do not cross-react with known p120 proteins. In one embodiment, the protein encoded by clone 188-28-4-0-B12-CS, a variant of said protein, and a bioactive fragment of said protein or said variant are nucleolar in nuclear fractionation studies. It can be used as a fractionation marker or a marker for preribosomal particles in methods known to those skilled in the art. In another embodiment, the protein encoded by clone 188-28-4-0-B12-CS, variants of said protein, and bioactive fragments thereof can be used as a proliferation marker in tumor cells. Alternatively, a quantitative immunoassay can be used to assess protein levels in excised cancerous and normal tissues. Alternatively, the protein levels encoded by clone 188-28-4-0-B12-CS can be compared between individuals and "normal" control groups as prognostic indicators of malignancy. Furthermore, the relationship between protein expression and cell proliferation can be assayed using cancer cell lines. Therefore, the protein of the present invention or a part thereof can be used as a proliferation marker in human cancer cells in vivo and in vitro. Confirmation of the absence of expression of the protein of the invention in normal and benign tumors serves as a marker for malignant cancer cells. Cancer cell growth rate is also
188-2 according to the method described by Trere D. et al. (J. Pathol. 192: 216-20, 2000).
It can be measured by quantitative analysis of the expression of the protein encoded by 8-4-0-B12-CS, or a biologically active fragment thereof.

The transforming activity of the proteins of the present invention was determined by Perlaky et al. (Anticancer Drug Des.
: 3-14, 1993). Therefore, (188-28-4-0-B12-
Polynucleotides encoding CS) proteins can be used to induce transformation in NIH / 3T3 cells in vitro. Alternatively, the polynucleotide encoding (188-28-4-0-B12-CS) can be used to provide antisense oligonucleotides useful in antisense therapeutic protocols according to methods known to those of skill in the art. <Protein of SEQ ID NO: 406 (Internal Designation No. 174-32-4-0-F8-CS)> The 378 amino acid long protein of SEQ ID NO: 406 encoded by the cDNA of SEQ ID NO: 165 is a protein of the colon, prostate, salivary gland, etc. It is expressed in tissues and overexpressed in the colon and prostate. The C-terminus of the protein of the present invention is a human retinoblastoma binding protein RbAp48 (Qian YW et al., (1993) Nature 364: 648-652, GenBank.
Accession number: X74262) homologous to mouse (GenBank accession number: Q60972)
) And its homologues conserved in other organisms, including C. elegans (GenBank accession number: AF116530). The protein of the invention also contains two internal WD repeat clusters (Prosite PS00678, 267-304 and 333-370 amino acid positions, respectively), structural motifs involved in protein interactions in signal transduction pathways and transcriptional regulation (Neer EJ et al. (1994) Nature 371: 297-300; Neer EJ et al., (1
996) Cell 84: 175-178). Retinoblastoma protein (Rb) is the retinoblastoma gene product. Deletion or inactivation of both Rb alleles is essential for the formation of human retinoblastoma in both genetic and sporadic forms (Benedict WF et al., (1983) Science 219
: 973-975). Loss-of-function mutations in the Rb gene also affect osteosarcoma, breast cancer, small cell lung cancer,
It is found in a number of other tumor types including bladder cancer, prostate cancer and soft tissue sarcoma (Bookstein R et al. (1991) Crit. Rev. Oncog. 2: 211-227). Introduction of wild-type Rb gene into cultured retinoblastoma cells suppresses cell growth and its tumorigenic potential in nude mice (Huang HJ et al. (1988) 242: 1536-1566). Expression of normal Rb protein in prostate, osteosarcoma, breast and bladder cancer cells also suppresses its tumor phenotype, thus establishing the Rb gene as a tumor suppressor (Weinberg RA (1991) Science 254: 1138-1146. Is outlined in).

The Rb gene product has been shown to be a nuclear phosphoprotein that undergoes cyclic phosphorylation and dephosphorylation during the cell cycle. Rb is dephosphorylated or “in the early G1 phase”
It is hypophosphorylated, becomes phosphorylated just before S phase, and remains phosphorylated until the latter half of mitosis. Injection of non-phosphorylated Rb protein into cells in early G1 phase
It suppresses entry into S phase, and thus some functions of Rb's growth suppressor are
suggest that this may be accomplished by the hypophosphorylated form of b (Goodrich et al.,
(1991) Cell 67: 293-302; Hinds PW et al., (1992) Cell 70: 993-1006). Rb proteins not only control the cell cycle, but are also involved in cell differentiation. For example,
Lens epithelial cells in Rb-deficient mice fail to undergo terminal differentiation and undergo apoptosis (Morgenbesser et al. (1994) Nature 371: 72-74). It has been demonstrated that the Rb protein suppresses cell growth and proliferation through its interaction with multiple cellular proteins that interfere with the downstream effects of these cellular proteins. For example, the Rb protein can form a specific complex with the transcription factor E2F, which regulates the expression of a series of genes essential for the G1 to S phase transition (Nevin JR et al.,
(1992) Science 258: 424-429). The Rb protein arrests cell cycle progression by masking the E2F transactivation domain or blocking the interaction of surrounding enhancer elements and the basal transcription complex (Weintraub SJ et al.,
(1995) Nature 375: 812-815). The association of Rb and the ribosomal transcription factor UBF results in inhibition of ribosomal RNA synthesis by RNA polymerase I (Cavanaugh
LI et al. (1995) Nature 374: 177-180; Mancini M et al. (1994) Proc. Natl. Ac.
ad. Sci. USA 91: 418-422). RbAp48 was first identified as a major protein from Hela cells that binds to a putative functional domain at the C-terminus of Rb protein. Only unphosphorylated and hypophosphorylated forms of Rb protein were co-precipitated with RbAp48. Similar to Rb protein, RbAp
48 is a ubiquitously expressed nuclear protein that shares sequence homology with MSI1, a negative regulator of the Ras-cAMP pathway in the yeast Saccharomyces cerevisiae. Rb
Overexpression of Ap48 is capable of converting the yeast mutant strain from heat shock sensitivity to heat shock resistance, which is similar to the results obtained from MSI1 overexpression. Thus, human RbAp48 is a functional homologue of MSI1 (Qian YW et al. (1993).
Nature 364: 648-652).

The Rbap48 protein is the p48 subunit of mammalian chromatin assembly factor 1 (CAF-1) and was later discovered to be present in the histone deacetylase complex (Parthum MR et al. (1996) Cell 87: 85-94). Human cell nucleus-derived CAF-1 is composed of three subunits, p150, p60 and p48, and is involved in the assembly of histone 3 and histone 4 on the nascent nucleosome structure during DNA replication in S phase (Kaufman FD et al., (1995) Cell 81: 1105-1114). Indeed, some transcriptional repressors function by recruiting the histone deacetylase complex (HDAC), the latter acting by acetylating or deacetylating the protruding tails from the histone core, thereby localizing chromatin. Modulate structure (Pazi
n MJ et al. (1997) Cell 89: 325-328). Rb protein is E2F
Suppresses transcription by recruiting HDACs to bind to (Brehm A et al. (1998) Natur
e 391: 597-601; Magnaghi-Jaulin L et al., (1998) Nature 391: 601-604). It was also reported that the chicken CAF-1 p48 subunit is capable of binding to chicken HDACs in vitro, which occurs through the interaction of WD-40 repeats present in both protein sequences (Ahmad A et al. (1999) J. Biol. Chem. 274: 16646.
-16653). The WD-40 protein family is characterized by loosely conserved repeats of approximately 40 amino acids, each repeat separated from each other by a Trp-Asp dipeptide sequence. The conserved core of this repeat, usually ending with the amino acid Trp-Asp (WD), was first identified in the beta subunit of the G protein, a heterotrimeric GTP-binding protein (Fong H et al., (1986). Proc. Natl. Acad. Sci.
USA 83: 2162-2166). All of the WD-40 proteins identified to date appear to have regulatory functions, not all enzymes (Neer, EJ et al. (1994) Nat.
ure 371: 297-300). Some WD repeat proteins are localized in the nucleus and function in repressing transcription. These include the Tup1, Hir1, and Met30 in nematodes; SCON2 in Drosophila; extra sex combs and Groucho in Drosophila; COP1 in Arabidopsis; and the family of HIRA and TLE proteins in humans. These WD-40 proteins are widely diverse in genes, including those involved in segregation, sex determination, and neurogenesis (controlled by Groucho), and those involved in photomorphogenesis (controlled by COP1). Shut off. It has been proposed that all of these WD-40, including proteins, fold into a surface-exposed propeller with a special loop to which the internal beta chain forms a rigid backbone to which other proteins bind. (Lambright DG et al. (1996) Nature 379: 31.
1-319; Sondex J et al. (1996) Nature 379: 369-374).

Thus, the discovery of new Rb-binding proteins is needed to design methods for blocking tumor development by controlling tumor growth and controlling tumor suppressor proteins in interacting with oncogene products. , Which is believed to provide new compositions that are useful in the diagnosis, prevention and treatment of cancer and developmental disorders. The protein of SEQ ID NO: 406 or a part thereof is used for controlling gene expression,
Probably involved as a transcriptional repressor. It is believed that the proteins of the present invention are capable of binding other proteins, preferably nuclear proteins, more preferably Rb. Preferred polypeptides of the invention are 159-373, 267-304.
And a polypeptide comprising a fragment of SEQ ID NO: 406 from positions 333-370. Other preferred polypeptides of the invention are polypeptides comprising a fragment of SEQ ID NO: 406 that has any of the biological activities described herein. The ability of the proteins of the invention, or portions thereof, to function as transcriptional repressors has been previously described (Weintraub SJ (1995) Nature 375: 812-815; Qian YW (1
995) J. Biol. Chem. 270 :: 25507-25513) and can be evaluated using techniques known to those skilled in the art. The ability of a protein of the invention or a portion thereof, particularly a fragment containing a WD repeat, to bind other proteins can be assessed using techniques known to those of skill in the art, including those described herein. For example, the protein of the invention is
As a "bait" protein in a hybrid system (Gal-4 system from Clontech), it is used to isolate as well as finally identify interacting in vivo protein partners from a cDNA library. Alternatively, the protein of the invention, or a portion thereof, may be used as a purified form or a fused form (which binds to a reporter gene product such as alkaline phosphatase), a phage cDNA expression library derived from a selected tissue or the cell type of a given organism. (Scott et al., (1990) Science 249: 386-390; Lam et al., (1992) Nature 354: 8.
2-84). Preferably, the binding capacity of the proteins of the invention is tested in mammalian cell transfection experiments. In an expression vector, the protein that binds to the expressed fusion protein when introduced into cultured cells in frame fusion with an appropriate peptide tag, such as <His> 6 in the pRset expression plasmid vector (Invitrogen), , Can be immunoprecipitated with anti- <His> 6 antibody. This method can also be used to confirm findings obtained either from the yeast two-hybrid system or in vitro screening of phage peptide libraries. In this case, the putative interaction partner proteins are fused to different tags in the secondary expression vector and cotransfected into the cultured cells. The true binding complex is co-immunoprecipitated with two different anti-tag antibodies. In certain embodiments, affinity chromatography methods are performed to identify in vitro partners of interacting proteins from cell lysates, as were done to identify RbAp48 proteins (Qian
YW et al. (1993) Nature 364: 648-652).

Embodiments of the present invention include a protein of the present invention or a portion thereof, particularly a poly containing a WD motif, for identifying and / or quantifying a binding protein, preferably a nuclear protein, more preferably Rb in a biological sample. Methods involving the use of peptides, or derivatives thereof, are therefore used in assays and diagnostic kits for the quantification of such binding proteins in body fluids, tissue samples, and mammalian cell culture. Such an assay may be particularly useful as a diagnostic or prognostic tool in the detection and monitoring of disorders associated with impaired expression of transcription factors. Such assays are therefore extremely useful for assessing levels of tumor suppressor Rb in disorders, including but not limited to cancers such as developmental disorders, retinoblastoma, prostate cancer, osteosarcoma, breast cancer and bladder cancer. It is useful. The binding activity of the protein of the present invention or a part thereof can be evaluated using any method known to those skilled in the art.
Preferably, a defined amount of the protein of the invention or part thereof is provided under conditions capable of forming a complex between the protein of the invention or part thereof and the binding protein to be identified and / or quantified. Add to sample. The presence of the complex and / or free protein of the invention or a portion thereof is then assayed and compared to controls using techniques known to those of skill in the art. Other embodiments of the invention relate to compositions and methods that employ the proteins of the invention, or portions thereof or derivatives thereof, to block in vitro or in vivo gene transcription. In a preferred embodiment, the protein of the invention, or a portion thereof or a derivative thereof, is added to the in vitro culture in an effective amount to suppress gene expression, thus cell growth using molecular biology techniques known to those of skill in the art. Enables the transfer of proteins from the extracellular medium to the cell nucleus. In another embodiment, the eukaryotic cell is a protein of the invention or a protein of the invention under certain conditions that prevents further growth upon demand such as infection, transformation, activation, differentiation, termination of the production process. It is genetically engineered to express a portion. Preferred embodiments of the present invention include cancer and hyperaldosteronism, adrenocortical hypofunction (Addison's disease), hyperthyroidism (Graves' disease), hypothyroidism, colorectal polyps, gastritis, gastric and duodenal ulcers, ulcers. Disorders associated with aberrant cell differentiation, proliferation, or degeneration, including systemic colitis, and Crohn's disease; gene transcription, such as metabolic diseases such as obesity and some inflammatory diseases due to interleukin overexpression A composition or method that uses SEQ ID NO: 406, SEQ ID NO: 165, or a portion thereof for the diagnosis, treatment, and / or prevention of disorders, including but not limited to disorders associated with dysfunction. For diagnostic and prognostic purposes, expression of the proteins of the invention may be determined by Northern blot, RT-PCR, as described herein.
, Can be studied using immunoblotting techniques and compared to expression in control individuals. For prophylactic and / or therapeutic purposes, expression of a protein of the invention may be
Overexpression can be achieved using any of the gene therapies known to those of skill in the art, including those described herein. For example, expression of a protein of the invention may be expressed in a retroviral or adenoviral vector that expresses the desired protein at high levels necessary to suppress mutations in the Rb gene or other oncogenes or tumor suppressor genes. It can be upregulated by infecting cells.

Other related embodiments relate to the use of SEQ ID NO: 406, SEQ ID NO: 165, their complements, or portions thereof to develop antagonists of the proteins of the invention.
These antagonists are antisense oligonucleotides, triple helices, ribozymes, small molecules or antibodies, especially neutralizing antibodies that bind to the WD repeats of the invention and are used in the treatment of diseases and conditions resulting from abnormally low transcription. obtain. These conditions include cocaine syndrome, accelerated aging syndromes such as ataxia telangiectasia and Werner syndrome, and age-related disorders, as well as "early onset" forms of age-related disorders such as dementia and Parkinson's disease. . In another embodiment, the invention relates to methods and compositions using the protein of the invention, or a portion thereof, as a marker protein for selectively identifying tissues, preferably colon and prostate. For example, the proteins of the invention, or portions thereof, can be used to synthesize specific antibodies using techniques known to those of skill in the art, including those described herein. Such tissue-specific antibodies can then identify tissue of unknown origin, such as differentiated tumor tissue that has metastasized to forensic samples, different body sites, etc., or use immunochemistry to identify different tissue types in tissue cross-sections. Can be used to classify. <Protein of SEQ ID NO: 414 (Internal Designation No. 188-27-3-0-G1-CS)> Expressed in brain, fetal brain, placenta and testis, overexpressed in brain, and SEQ ID NO: 17
The 389 amino acid long protein of SEQ ID NO: 414, encoded by the cDNA of 3, is SIRTUIN-2.
(SIRT2) (Trembl accession number: Q9Y6E9) and silent information control factor type 2 (
Silent information regulator 2-like protein (SIR2L) (Tre
mbl Accession number: Q9UNT0) Furthermore, the protein of the invention is SIR2
Presents the Pfam signature of the family members (amino acids 84-268). Furthermore, the proteins of the invention have been shown to be essential for SIR2 silencing function,
It exhibits two characteristic motifs that are highly conserved in all SIR2 family members (Moira M. et al., Genetics, 154: 1069-1083 (2000)). These motifs are from positions 84-98 and 165-170 of the protein of SEQ ID NO: 414, and G
Corresponds to AG (I / V) SxxxG (I / V) PDFERS and (Y / I) TQNID patterns respectively. The protein of SEQ ID NO: 414 also has a DNA binding Zn finger motif (Prodom putative number
PD002659, 195-224 positions) or having conserved cysteine residues at positions 195, 200, 221, and 224, which cover any possible enzyme domain (or enzyme cofactor) (Moira M. et al., Genetics, 154). : 1069-1083 (2000))
.

The cDNA of SEQ ID NO: 173 encoding the protein of the invention differs from the cDNA encoding the SIRT2 protein by the complement exon located between positions 147 and 195. This exon modifies the start codon of the protein, extending the ORF to its N-terminal portion by 16 amino acids. In addition, amino acid residues at positions 20 and 21 of the protein of the invention (alanine and glutamine residues, respectively) have been replaced with glutamine and tyrosine residues (positions 4 and 5) of the SIRT2 protein. Thus, the protein of the invention is a new isoform of SIR2 due to alternative splicing. The protein of the present invention of SEQ ID NO: 414 also has its N-terminus 37 amino acids longer than the SIRT2 protein. Regulation of gene expression by modification of chromatin structure is a universal mechanism in eukaryotic cells and responsible for maintaining gene expression patterns throughout the development of multicellular organisms. Silencing has been studied most closely in S. cerevisae (yeast). Among the SIR genes, SIR2 is the most evolutionarily conserved and SIR2
Several genes have been identified that have homology to Frye R et al., Biochem.
Ophys. Res. Commun., 273: 793-798 (2000)). The presence of homologues of SIR2 (HST) in organisms from bacteria to humans suggest that the silencing mechanism of SIR2 may be conserved. It was initially discovered that SIR2 influences zygotic control in monophasic somatic cells by locus-specific transcriptional silencing. SIR2 and its homologues have been suggested to play additional roles in recombination, chromosomal stability, metabolic regulation, suppression of meiosis and senescence (for a review, see
Gartenberg, Curr. Opin. Microbiol. 3: 132-137 (2000)). The SIR2 family of proteins are also considered to be either enzymes or enzyme cofactors. First, Landry and coworkers show that members of the SIR2 family catalyze histone deacetylation in reactions requiring NAD, thereby distinguishing it from previously characterized deacetylases. It was This enzyme is active on histone substrates acetylated by both chromatin assembly-related and transcription-related acetyltransferases (Landry et al., Proc. Nat.
l. Acad. Sci. 97: 5807-5811 (2000)). The discovery of the endogenous deacetylation activity of the conserved SIR2 family provides a mechanism to modify histones and other proteins to regulate transcription and diverse biological processes. Next, human SI
Studies of R2 family members (hSirT2) were found to have mono-ADP ribosylation activity in vitro (Frye R et al., Biochem. Biophys. Res. Co.
mmun., 260: 273-279 (1999)). Potential substrates for mono-ADP ribosylation include histones and RNA Pol whose modifications correlate with enhanced rDNA transcription.
I have.

The protein of SEQ ID NO: 414, or a portion thereof, is probably a member of the SIR2 protein family and is used for gene silencing, repression of recombination, chromosomal stability,
It is believed to be involved in metabolic regulation, meiosis, and aging. In particular, the protein of the present invention can deacetylate substrates, preferably acetylated histones and acetyltransferases, directly or indirectly as enzyme cofactors. In particular, the proteins according to the invention are preferably linked to the ribosylating activity, preferably mono-AD, as an enzyme cofactor, preferably in histones and RNA Pol I substrates.
May have P-ribosylation activity. Further, the protein of the present invention may be a DNA binding protein. Preferred polypeptides of the invention are 1-16, 84-98,
A polypeptide comprising the amino acid of SEQ ID NO: 414 from positions 165-170, 195-224 and 84-268, and SEQ ID NO: comprising at least one cysteine residue located at position 195, 200, 221 or 224 of SEQ ID NO: 414. It is a fragment of 414. Other preferred polypeptides of the present invention are fragments of SEQ ID NO: 414 having any of the biological activities described herein. The deacetylation activity of a protein of the invention, or a portion thereof, can be assayed using any of the assays known to those of skill in the art, including those described in Laundry et al. (Supra). Ribosylation activity of the proteins of the invention or portions thereof can be assayed using any of the assays known to those of skill in the art, including those described in Frye et al., (1999). Nucleic acid binding activity of the proteins of the invention or portions thereof can be assayed using any of the assays known to those of skill in the art, including those described in US Pat. No. 6,013,453. The present invention relates to methods and compositions using the proteins of the invention, or portions thereof, for silencing gene expression. In a preferred embodiment, an amount of a protein of the invention, or a portion thereof or a derivative thereof, that is effective in suppressing gene expression is added to the in vitro culture, and thus cell growth using molecular biology techniques known to those of skill in the art It enables the transfer of this protein from the outer medium to the cell nucleus. In other embodiments, the eukaryotic cells may be treated under certain conditions, such as upon infection, transformation, termination of the production process, differentiation, etc., under certain conditions to prevent further proliferation of such cells. It is genetically engineered to express the protein or a portion thereof.

The present invention relates to methods and compositions that use the proteins of the invention or portions thereof to deacetylate a substrate, either alone or in combination with other substrates. Such substrates are acetylated substrates, preferably acetylated histones and acetyltransferases. For example, a protein of the invention or a portion thereof is added to a sample containing substrate (s) under conditions that allow deacetylation and catalyze the deacetylation of substrate (s). To be done. In a preferred embodiment,
Deacetylation is performed using standard assays such as those described in Laundry et al., Supra. Deacetylated histones obtained by this method can be mixed with purified naked DNA (eg plasmid preparations) to reconstitute a chromatin-like structure in vitro. Such structures are of great interest in the study of enzymatic factors involved in transcription and replication. Other embodiments of the invention include in vitro screening of inhibitors directed against encoded deacetylase activity using any technique known to those of skill in the art, including those described herein. Compositions and methods of using the proteins of the invention or portions thereof to develop an assay for use. Such deacetylase inhibitors have great potential as new drugs because of their ability to influence transcriptional regulation as well as induce apoptosis or differentiation in cancer cells. Preferably, proteins of the invention expressed in prokaryotic or eukaryotic systems are mixed in vitro with a simple fluorescent substrate, such as an aminocoumarin derivative of acetylated lysine, and different putative inhibitors, according to methods known to those skilled in the art. do it. The coumarin derivative is then quantified with a fluorescence detector using a reverse phase HPLC system. Such methods have been previously developed by Hoffmann and coworkers (Hoffmann et al., Nucl Acids Res. 27: 2057-2058 (1999); Hoffmann et al.
Pharmazie. 55: 601-606 (2000)). The present invention relates to methods and compositions, which alone or in combination with other substances, use a protein of the invention or a part thereof for binding a nucleic acid, preferably DNA. For example, a protein of the invention or a portion thereof is added to a sample containing nucleic acid under conditions that allow binding as well as binding to the nucleic acid. In a preferred embodiment, the proteins of the invention or portions thereof can be used to purify nucleic acids such as restriction fragments. In another preferred embodiment, the proteins of the invention or portions thereof can be used to visualize nucleic acids as the polypeptide binds to the appropriate fusion partner or as detected by antibody search. Alternatively, the protein of the invention, or a portion thereof, may be bound to a chromatographic support using techniques known in the art, either alone or in combination with other DNA binding proteins, to make affinity chromatography columns. it can. The sample containing the nucleic acid to be purified is passed through the column. Immobilizing the protein of the invention or a portion thereof on a support is particularly advantageous for embodiments in which the method is practiced on a commercial scale. This immobilization facilitates removal of the protein from the batch product and subsequent reuse of the protein. Immobilization of the protein of the invention or a part thereof can be achieved, for example, by inserting a cellulose binding domain into the protein. One of ordinary skill in the art will appreciate that other methods of immobilization are also available and are described in the published literature.

Yet another embodiment of the present invention is the use of a protein of the invention or part thereof to identify a gene or region of the human genome which is silenced by the protein of the invention or part thereof. Compositions and methods for use. Genome DN derived from patients with pathological conditions such as cancer and metabolic disorders, or the elderly
A is compared to the genomic DNA extracted from each control for its ability to bind the protein of the invention. As explained above, the protein of SEQ ID NO: 414 is
It presents a putative Zn finger domain capable of binding to DNA sequences near the silencing region or gene. The proteins of the invention, or portions thereof, can be attached to chromatographic supports to create affinity chromatography columns using techniques known to those of skill in the art. A sample containing a mixture of human genomic DNA digested with endo-restriction enzymes is passed through the column. After careful washing, the bound DNA is eluted and further subcloned into conventional cloning vectors known to those of skill in the art. Immobilizing the protein of the invention, or a portion thereof, on a support is particularly convenient for embodiments in which the method must be routinely performed. This immobilization facilitates the removal of DNA from the batch of protein bound protein after conjugation, allowing the protein to be reused later. Immobilization of a protein of the invention or a portion thereof can be accomplished, for example, by inserting a matrix binding domain into the protein according to methods known to those of skill in the art. The resulting fusion product containing the protein of the invention or a portion thereof is then covalently or otherwise attached to the protein, carbohydrate or matrix (gold, "Sephadex" particles, and polymer surface). It Another embodiment of the invention relates to a method of preparing an antibody directed against a protein of the invention or a portion thereof. Such antibodies can be used in co-immunoprecipitation methods to enrich chromatin fragments containing the binding site for the proteins of the invention. This method can identify genes or human genomic regions that have been silenced by the deacetylation activity of the proteins of the invention. Preferably, in a sample containing intact chromatin fragments, the antibody directed against 414 and bound to protein A or protein G sepharose beads is added to the mixture. Immunoprecipitation conditions are known to those of skill in the art. After washing, the DNA fragment coprecipitated with 414 is extracted and further subcloned into routinely used cloning vectors. These DNA fragments can be sequenced,
And / or used as a probe for genomic library screening. This method is very similar to that used by Gould and coworkers to enrich embryonic chromatin fragments containing homeotic Ubx protein sites (Gould et al., Nature 348: 308-312 (1990)).

A preferred embodiment of the present invention is a composition that uses SEQ ID NO: 414, SEQ ID NO: 173, or a portion thereof for the diagnosis, treatment, and / or prevention of developmental disorders that result from the expression of "disease-causing" genes. Or regarding the method. The number of conditions and conditions treatable by the proteins of the invention can be enormous and infinite. Convenient disorders include cancer and hyperaldosterone, hypoadrenocorticism (Addison's disease), hyperthyroidism (Graves' disease), hypothyroidism, colorectal polyps, gastritis, gastric and duodenal ulcer, ulcerative Colitis, and other disorders associated with abnormal cell differentiation, proliferation or degeneration, including Crohn's disease; viral infections, particularly HIV and viral hepatitis (ie viral protein expression), metabolic diseases such as obesity and interleukin overexpression It is associated with dysfunction of gene transcription such as some inflammatory diseases caused by. For diagnostic purposes, expression of the proteins of the invention can be studied using the Northern blotting, RT-PCR, immunoblotting methods described herein and compared to expression in control individuals. For prophylactic and / or therapeutic purposes, the proteins of the invention can be overexpressed using any of the gene therapy methods known to those of skill in the art, including those described herein. For example, blocking a "disease" gene
For example, this can be achieved by directly targeting the protein of the present invention or a part thereof to silence the expression (silencing) of an overexpressed gene (such as an oncogene in cancer). This is the "chimera" in which the putative Zn binding domain is replaced by a sequence known to bind it, or a sequence in the vicinity of the overexpressed gene, as described elsewhere in this application. It can be achieved by creating a protein. Deacetylase activity and specific DN
Fusion proteins containing both A-binding domains can be obtained by methods of molecular biology known to those skilled in the art. In the case of cancer, metabolic disorders, aging and disorders in which the gene is overexpressed, the corresponding eukaryotic expression vector can be used for gene therapy. Such recombinant cDNAs can be introduced into the famous adenovirus vectors used in cancer therapy (for a recent review on the use of replicative adenoviruses for cancer therapy: Alemany et al., Nat. Biotechnol. 18: 723. -727 (20
00)). Other related embodiments relate to the use of SEQ ID NO: 414, SEQ ID NO: 173, complements thereof, or portions thereof to develop antagonists of the proteins and SIR complexes of the invention. These antagonists are antisense oligonucleotides, triple helices, ribozymes, small molecules or antibodies and can be used to treat diseases and conditions resulting from abnormal gene silencing. These conditions include cocaine syndrome, accelerated aging syndromes such as ataxia telangiectasia and Werner syndrome, and age-related disorders, as well as "early onset" forms of age-related disorders such as dementia and Parkinson's disease. .

In another embodiment, the present invention relates to methods and compositions using the protein of the present invention or a portion thereof as a marker protein for selectively identifying tissue, preferably brain tissue. For example, the proteins of the invention, or portions thereof, can be used to synthesize specific antibodies using techniques known to those of skill in the art, including those described herein. Such tissue-specific antibodies can then identify tissue of unknown origin, such as differentiated tumor tissue that has metastasized to forensic samples, different body sites, etc., or use immunochemistry to identify different tissue types in tissue cross-sections. Can be used to classify. <The protein of SEQ ID NO: 298 (internal designation number 182-1-2-0-D12-CS)> The protein of SEQ ID NO: 298 encoded by the cDNA of SEQ ID NO: 57 is a protein of the fibroblast growth factor family (FGF). Is homologous to. In particular, SEQ ID NO: 298
The amino acid sequence of is similar to the recently described FGF-23. The protein of the present invention is strongly expressed in fetal liver. The protein of the invention exhibits the pfam signature of fibroblast growth factor (position 48-129). High-resolution X-ray crystal structures of both FGF-1 and FGF-2 have been reported and linked to form a triple symmetric structure composed of four-stranded antiparallel beta sheets.
Revealed a "beta-trefoil" morphology containing two chains (Faham S. et al.,-Curr. Opi
n. Struct. Biol.-1998, 8 (5): p578-586). Based on sequence conservation, FGF
It is very likely that all members of the family have related three-dimensional structures. Preferred polypeptides of the invention are 39-45; 51-56; 60-64
71-77; 82-87; 92-97; 101-105; 113-119; 124-130; 142-147
Containing amino acids at positions 151-155 and / or 167-172, which, by homology, are characteristic of the FGF family with other members of the FGF family
Produces 12 beta pleated sheets (White K. et al.-Nat. Genet.-2000; 26
(3): pp. 345-348). Furthermore, because in some of these regions some amino acids from SEQ ID NO: 298 are conserved for 80% of human FGF (after sequence alignment), the most preferred polypeptides of the invention are amino acids 42, 53, 63, 83, 85
, 87, 93, 96, 101, 113, 115, 124, 127 and / or 129. Other preferred polypeptides of the present invention are fragments of SEQ ID NO: 298 having any of the biological activities described herein.

Cytokines are a heterogeneous group of polypeptide mediators that are associated with numerous functions, including the immune system and inflammatory responses. The cytokine family includes, but is not limited to, interleukins, chemokines, tumor necrosis factors, interferons, colony stimulating factors, neurotrophins, neuropoietins and growth factors, of which the FGF family is a member. Fibroblast growth factor (FGF) was first characterized in the mid-1970s as a mitogen for cultured fibroblasts. Since then, over 20 different FGFs have been identified. Fibroblast growth factor belongs to a family of proteins called growth factors (other members of this family are EGF, PDGF, TGF and EC.
Including GF). The biological effects of FGFs are mediated by their association with three biochemically distinct partners, which are heparan sulfate proteoglycans, low affinity transmembrane FGF binding proteins and high affinity transmembrane FGFs of the tyrosine kinase class. It is a receptor. Transfection and reconstitution experiments showed that intracellular signaling was FG
It was shown to be triggered by activation of F receptor kinase activity. Activation probably results from receptor oligomerization mediated by the association of heparan sulfate proteoglycans and ligands (FGF), and ligands and the receptors themselves (Faham S. et al.,-Curr Opin Struct Biol.-1998. , 8 (5): p578-58
6). Long heparin-derived oligosaccharides generally exhibit close binding to FGF. The relationship between heparin length, biological activity, and FGF binding has been extensively studied and it is generally accepted that longer length heparin oligosaccharides tend to have higher biological activity. FGF is a member of a family with a wide range of biological activities involved in cell growth and differentiation (including angiogenesis, morphogenesis and wound healing), cell survival, replication, adhesion and motility. FGF may be a potent growth factor for several cell types including but not limited to fibroblasts, endothelial cells, smooth muscle cells, keratinocytes (keratinocytes), osteoblasts and neurons. Have been discovered. Clearly, FGF biology can be extremely complex, involving multiple ligands, receptors and cofactors, each expressed with distinct spatial and temporal patterns and unique kinetics during normal development. is there. There are tremendous efforts to create different types of animal models for in vivo FGF function analysis.
These studies clearly indicate that FGF signaling is involved in several different processes at different stages of development and is important during early developmental stages (FGF-4 and FGF receptor 1 homozygous). Both sex null mutations are lethal). FGF signaling has been found to be required for the establishment of a normal program of lung branching morphogenesis and cutaneous keratinocyte differentiation. FGF signaling has also been found to be involved in the early induction of limb bud growth and its continued growth during early limb development. Perhaps the most striking illustration of this function of FGF signaling is its ability to induce chick excess limb development by topical administration of FGF-soaked beads (Cohn M et al.-Cell-1995; 80: p739-746). , Thus indicating that at least some FGF-dependent processes are controlled by accessibility to FGF ligands. FGFs are also able to stimulate migration and differentiation of liver precursors.

Recently, a mutation in the FGF-23 gene is a genetically transmitted disease characterized by low serum phosphate levels, rickets, osteomalacia, leg deformities, short stature, bone pain, and tooth abscesses Was found to be associated with autosomal dominant hypophosphatemic rickets (ADHR). FGF signaling functions are involved in many aspects of morphogenesis, differentiation and other essential cellular mechanisms, and are therefore most likely involved in many diseases and conditions associated with these processes. Be done. Thus, the protein of SEQ ID NO: 298 is a member of the fibroblast growth factor family and thus includes cell growth and differentiation, angiogenesis, morphogenesis, wound healing, cell survival, replication, adhesion and motility, It is believed to be involved in numerous cellular and biological processes including, but not limited to. One embodiment of the invention relates to the use of the polypeptides and polynucleotides of the invention to identify liver, heart, thyroid and parathyroid cells, or cells derived from these tissues, because This is because the protein of the invention is expressed there (White K. et al.,-Nat Genet.-2000; 26 (3): p345-348.
). Such detection of cells expressing the protein is accomplished using standard antibodies or antisera raised against the protein using standard methods, as well as polynucleotides specific for the nucleic acids encoding the proteins of the invention. This can be done in any of several ways, including using a probe. In other embodiments, the proteins of the invention, or portions thereof, induce growth of several different cell types, including but not limited to fibroblasts, myocytes, osteoblasts, keratinocytes and hepatocytes. Can be used as a mitogen to stimulate. Cell growth can be stimulated in vitro, for example, to promote growth of cells that are cultured for recombinant protein synthesis or for ex vivo gene therapy applications. Other preferred applications of the present technology include the proteins of the invention, or portions thereof, for producing tissues and organs in vitro, including but not limited to skin, cartilage, and bone for transplants and implants. Regarding use. Another preferred embodiment of the invention relates to the use of the protein according to the invention or a part thereof for treating damaged tissues and organs. Members of the FGF family have been shown to induce the differentiation and growth of several different cell types. Thus, the proteins of the invention can be administered to treat pathologies and conditions that result from damage to cells, tissues or organs. These pathologies and conditions are associated with fractures and bone defects (Kimoto et al.,-J Dent Res.-1998, 77 (12): p1965-1969) (Sol
heim E-Int Orthop,-1998, 22 (6)), wound-induced damage (lesions of skin and ulcer, etc.) (Debus E.-Zentralbl Chir.-2000, 125 (supple 1): p49-55
) (Szabo S.-Aliment Pharmacol Ther.-2000, 14 (Suppl 1): p33-43),
Tissue damage due to ischemia (eg brain and heart) (Simons M.-Circulatio
n-2000, 102 (11): pE73-E86), cardiovascular diseases such as thrombosis and atherosclerosis (Bauters C.-Drugs-1999, 58 (Spec No1): p11-15), and Parkinson's disease. Or neurodegenerative diseases caused by neurological loss such as Alzheimer's disease (Ebadi M-Neurochem Int.-1997, 30 (4-5): p347-374) (Brundin P.-
Cell Transplant.-2000, 9 (2): p179-195), but is not limited thereto.

In a most preferred embodiment, the polypeptides or polynucleotides of the invention are non-functional, such as autosomal dominant hypophosphatemic rickets, associated with mutations of specific amino acids of FGF-23. And / or diagnose a disease caused by a mutated FGF,
Can be used to treat or prevent (White K. et al.,-Nat Genet.-2000;
26 (3): p345-348, the entire text of which is incorporated herein by reference).
Such disorders can be treated, for example, by administering a therapeutically effective amount of a protein of the invention or a polynucleotide sequence encoding the protein to a patient suffering from the disease. Similarly, SEQ ID NO: 298 or SEQ ID NO: 57, or a portion thereof, may be used to diagnose, prevent, and / or treat other FGF-related disorders, such as conditions associated with FGF overexpression. Can be used to develop kits. In yet another embodiment, the protein of the invention, or a portion thereof, is used to treat a disorder associated with FGF and And / or can be used to develop antagonists of the FGF receptor. This is especially true for pathologies such as cancers in which some tumors secrete large amounts of FGF, such as prostate and breast cancer. Moreover, FGF antagonists are useful in inhibiting tumor angiogenesis, an essential step in tumor growth. Similarly, SEQ ID NO: 57, or a portion thereof, can be used to produce antisense oligonucleotides. Antisense oligonucleotides block complementary mRNAs and thus inhibit the synthesis of the protein encoded by the mRNA. These oligonucleotides can be used for in vivo or ex vivo treatment of diseases caused by or exacerbated by FGF overexpression. <Protein of SEQ ID NO: 396 (internal designation number 160-12-1-0-D10-CS)> Encoded by the cDNA of SEQ ID NO: 155 and overexpressed in the brain and fetal brain,
The protein of SEQ ID NO: 396 is the transmembrane superfamily of proteins 4 (TM4SF)
Shows homology to members of. The protein of the invention has a signature characteristic of this family: the pfam domain from positions 66-273, Prosi from positions 112-134.
te motif and 108-127, 108-146, 129-150, 128-154 and 247-274
Present the emotif domain from the location. In addition, the protein of the invention has several predicted transmembrane domains 103-123, 130-150 and 245-265, with less certainty but additional predicted domains from positions 61-81. Have. The proteins of the invention, except for their N-termini, have significant homology to TM4SF members, a complex membrane CD81 antigen also known as TAPA1 (antiproliferative antibody). The transmembrane domain of the protein of the present invention matches that described for CD81.

Members of the tetraspan family of proteins associate with adhesion molecules and translate adhesive events into controls of cellular behavior. TAPA-1 is a widely expressed protein that has been found to affect adhesion, morphology, activation, proliferation and differentiation of B, T and other cells. TAPA-1 has two long hydrophobic domains that are extracellular and also consist of molecules located between four TMs (transmembrane regions, TM1-4). TM
The region between 2 and TM3 is highly conserved in all tetraspanins. The protein is highly hydrophobic and contains a latent N-myristoylation site. TAPA1 functions by forming a complex on the cell surface, and the antigenic epitope of human TAPA1 is contained within the subregion of the second extracellular domain of this protein. Cell surface expression of TAPA1 can be down-modulated by antibody binding (Levy 1991, J Bi
ol Chem. Aug 5; 266 (22): 14597-602). Mice lacking CD81 (not expressing TAPA1) have impaired antibody response to protein antigens. This defect is specific for antigens that preferentially stimulate the helper T2 response and are only observed as T cell-dependent antigens. The absence of CD81 on B cells certainly results in a defect. The production of antigen-specific interleukin (IL) 4 is
It is significantly reduced in the spleen and lymph nodes of CD81 null mice compared to their heterozygous littermates. Expression of CD81 on B cells is essential for induction of optimal IL-4 and antibody production during the helper T2 response. CD81 appears to have a more important role in controlling the immune response than in the development of immune cells (Maecker (1997)
J Exp Med 1997 Apr 21; 185 (8): 1505-10). CD81 on B cells is IL-derived from T cells.
4 Has the ability to promote secretion. Co-stimulatory proteins such as B7-1 and B7-2 have been shown to have specific effects on cytokine secretion by T cells in certain systems. CD81 on B cells can control cytokine production by T cells. TAPA1 is implicated in playing an important role in controlling lymphoma cell growth. TAPA-1 is highly expressed in many neurons of the brainstem. TAPA is found in all glial cells and the level of this protein correlates with its maturation (Sull
ivan et al., 1998, J Comp Neurol 1998 Jul 6; 396 (3): 366-80). This protein is expressed by the ependyma, choroid plexus, astrocytes, and oligodendrocytes. TAPA1 is dramatically upregulated during early postnatal development, when glial is born and matures. On the 18th day of embryonic day, TAPA level becomes lower,
It has most of the immune reaction products associated with the choroid plexus and glial boundaries. The amount of TAPA expressed in the brain increases with the development of the brain, and the protein level reaches the adult level at 14 days of age. This increase in TAPA levels at postnatal day 14 is due to upregulation in gray matter and white matter. TAPA is associated with reactive gliosis and glial scarring. CD81 with reactive microglia and astrocytes
And the spatiotemporal expression pattern of CD81 indicates that CD81 is involved in the glial response to spinal cord injury. Upregulation of TAPA has been suggested to be a complex component of glial scar formation (Peduzzi et al. Exp Neurol. 1999 Dec; 160 (2): 460-8).

TAPA-1 levels are low in metastatic prostate tumors, and expression of this protein in these cells appears to suppress metastatic behavior (Dong et al. 1995 Scie.
nce.12; 268 (5212): 884-6). Divalent antibodies directed against these proteins have been demonstrated by different cell types of pre-B cells (Masellis-Smith and Shaw (1994) J Immunol. 1).
994 Mar 15; 152 (6): 2768-77), endothelial cells (Forsyth, 1991 Immunology Feb;
72 (2): 292-6) and tumor cell motility and invasiveness (Miyake et al., 199).
1 J Exp Med. Dec 1; 174 (6): 1347-54). In the nervous system, Schwann cell migration to biologically relevant substrates is enhanced by administration of antibodies directed against specific TMs (Anton et al., (1995) JN.
eurosci Jan ； 15 (1 Pt 2): 584-95). Antibodies directed against TAPA-1 suppress mitotic activity and induce increased cell adhesion (Oren et al., 1990 Mol Cell Biol Aug.
10 (8): 4007-15). The protein of SEQ ID NO: 396, or a portion thereof, is believed to be involved in cell adhesion, motility, metastasis, cell activation, signal transduction and immune response, presumably as a member of the TM4SF family. As a member of the tetraspanin family of proteins, the protein of SEQ ID NO: 396, or a portion thereof, mediates cell interactions in lymphocytes as well as non-vascular lymphoid tissue to promote cell adhesion and migration,
It is believed to affect the modification of cell morphology as well as the activation state of cells. Preferred polypeptides of the invention are 66-273, 112-134, 108-127, 108-146, 129-
A polypeptide comprising amino acids of SEQ ID NO: 396 from positions 150, 128-154, and 247-274. Other preferred polypeptides of the present invention are fragments of SEQ ID NO: 396 that include any of the biological activities described herein. The activity of the proteins of the invention, or portions thereof, describes a functional tissue assay used to define surface antigens that control astrocyte growth (Eldon et al., 1996, J Neu.
rosci, 16 (17): 5478); Cell functional assay to determine the involvement of proteins in signal transduction and cell adhesion in the immune system (Levy et al. 1998 Ann. Rev. Immu.
Nol. 16: 89-109, Virtaneva et al., 1994 Immunogenetics 39: 329-334), and can be assayed using any of the assays known to those of skill in the art. An embodiment of the present invention is a membrane protein in a biological sample, preferably an integrin,
Methods of using the proteins of the invention or portions thereof to identify and / or quantify lineage-specific molecules, tetraspanins, and antibodies, and therefore to quantify such membrane proteins in tissue samples, and mammalian cell cultures. Used in assays and diagnostic kits. The binding activity of the protein of the present invention or a part thereof is determined by Shoshana et al., 1998, Annu. Rev. Immunol 16: 89-109; Maecker et al., 1998.
PNAS 95: 2458-2462; Geisert et al., 1996, J of Neuroscience 16 (17): 5478-.
It can be assessed using the assay described in 5487, or other methods known to those of skill in the art. Preferably, a defined amount of the protein of the invention or a portion thereof is added to the sample under conditions that allow complex formation between the protein of the invention or a portion thereof and the membrane protein to be identified and / or quantified. To do. The presence of the complex and / or free protein of the invention or a portion thereof is then assayed and compared to controls using techniques known to those of skill in the art.

In another embodiment, the invention relates to compositions and methods of using the proteins of the invention or portions thereof to stimulate cell proliferation, preferably lymphocyte proliferation in vitro and in vivo. For example, an effective amount of a protein of the invention or a soluble form of a portion thereof may be added to the cell culture medium to stimulate cell proliferation. In other embodiments, the invention relates to compositions and methods that employ the proteins of the invention or portions thereof or derivatives thereof to stimulate antibody production in vitro or in vivo. In a preferred embodiment, an effective amount of a protein of the invention or a portion or derivative thereof to stimulate antibody production may be added during in vitro culture of antibody producing cells such as hybridomas. In another preferred embodiment,
In the case of polyclonal antibody production, the protein of the present invention or a part thereof or a derivative thereof is injected into an animal in order to increase the antibody production of the animal against the target protein. In yet another embodiment, the invention relates to compositions and methods that employ the proteins of the invention or portions thereof or derivatives thereof to reduce cell adhesion, either in vitro or in vivo. In a preferred embodiment, an effective amount of the protein of the present invention or a part thereof or a derivative thereof for preventing and / or suppressing cell adhesion is added during the in vitro culture of adherent cells in order to recover those adherent cells. it can. In yet another embodiment, the invention provides for cell proliferative disorders such as cancer, preferably brain cancer, via metastasis prevention and also autoimmune diseases, AIDS, allergies,
Diabetes mellitus type 1, systemic lupus erythematosus, rheumatoid arthritis, juvenile rheumatoid arthritis, Sjogren's syndrome, systemic sclerosis, mixed connective tissue disease and dermatomyositis, Hashimoto's disease, primary myxedema, thyrotoxicosis, pernicious anemia, ulcer Colitis, autoimmune atrophic gastritis, idiopathic Addison's disease, male infertility, Good Basture syndrome, acute progressive glomerulonephritis, myasthenia gravis, polymyositis, pemphigus, pemphigoid, sympathetic Such as ophthalmitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, post-myocardial infarction syndrome, rheumatic fever, lupus-like hepatitis, primary biliary cirrhosis, Behcet's syndrome and Crest syndrome, A protein of the present invention or a protein thereof, for treating and / or preventing a disorder characterized by a suppressive immune response via stimulation of antibody production and IL-4 secretion. In part, it relates to compositions and methods.

In another embodiment, the present invention relates to methods and compositions using the protein of the present invention or a portion thereof as a marker protein for selectively identifying tissues, preferably brain and fetal brain-derived tissues. . For example, the proteins of the invention, or portions thereof, can be used to synthesize specific antibodies using techniques known to those of skill in the art, including those described herein. Such tissue-specific antibodies can then be used to identify tissues of unknown origin, such as differentiated tumor tissues that have metastasized to forensic samples, different body sites, etc., using immunochemistry or other techniques known to those of skill in the art. Can be used to classify different tissue types in a tissue cross section. <Protein of SEQ ID NO: 296 (Internal designation 181-3-3-0-B8-CS)> The protein of SEQ ID NO: 296 encoded by the cDNA of SEQ ID NO: 55 is overexpressed in fetal liver, and C. elegans ( Caenorhabditis elegans), mouse and human well conserved (accession numbers Q06561, Q05793 and P98160 respectively) basement membrane specific heparan sulfate proteoglycan core proteins (perlecan) IV-4 and
It is homologous to all IV-5 domains. The 247 amino acid long protein of the invention has position 44
~ 64 and 219-239 two putative hydrophobic extensions and a putative immunoglobulin domain homologous to Ig domain 4 of the Ig repeat structure of domain IV of the perlecan protein is shown at positions 141-197. . The above protein of the present invention also comprises
Positions 6-21 show putative secretory signal peptides. The basement membrane is a specialized region of the extracellular matrix (ECM) that consists of a number of different components, including laminin, collagen, nidogen and heparan sulfate proteoglycans (for research, Bernfield at al., Annu. Rev. Biochem. 68: 729
-777 (1999)). Rev. perlecan is the major basement membrane and plays an important role in a number of underlying developmental and regenerative processes, including cell aggregation, adhesion and migration, signaling and gene regulation (Martin and Timpl, Annu. .68: 729-77
7 (1999)). Rev. Cell Biol. 3: 57-85 (1987)). The perlecan cDNA sequence encodes a large core protein consisting of five structural domains called IV, which are SEA modules (domain I), LDL class A modules (domain II), cysteine rich modules ( It has characteristic motifs such as domain III) and LAMB module (domain V). Domain IV consists of Ig-like repeats (14 in mouse and 21 in human perlecan) that are similar to the Ig-like repeats of neural cell adhesion molecule (N-CAM). Glycosaminoglycan chains are predominantly linked to domain I and have been shown to be involved in their differential expression of core proteins at organizational and developmental stages (Perrimon and Bernfield, Nature 404: 725-72).
8 (2000)).

The N-terminal fragment IV containing 1 to 8 Ig modules binds with high affinity to two known nidogen isoforms, laminin-1-nidogen-1 complex (LN), and We show that it binds to heparin at physiological ionic strength (Hop
f et al., Eur. J. Biochem. 259: 917-925 (1999)). Studies on C. elegans perlecan alterations in vivo show that mutations in Ig-like modules 3 and 4 inhibit the spatial distribution of splice variants and induce a lethal phenotype. Mutations that induce a deletion in other Ig-like modules of perlecan domain IV do not affect the expression and spatial distribution of the isoform, so that Ig-like modules 3 and 4 are critical for muscle formation and growth. Can be presumed to play a role (Mullen et al., Mol. B.
iol Cell 10: 3205-3221 (1999)). Several studies have shown that there are many different perlecan isoforms in C. elegans due to alternative splicing (Rogalski et al., G.
enes Dev. 7: 1471-1484 (1993), Rogalski et al., Genetics 139: 159-169 (1995)).
. Although splice variants have not been shown in humans, Ig-like modules are encoded by multiple exons that are compatible with a variety of possible expression combinations (C
ohen et al., PNAS 90: 10404-10408 (1993)). Alternative splicing in domain IV has been linked to temporal and spatial differences in isoform expression.
A subset of C. elegans isoforms are associated with body wall muscle during embryogenesis and are required for linear animal myofilament lattice assembly, which is very similar to focal adhesion formation in mammalian cell cultures. (Moerman and Fire, CSH labo.
Press (1997)). Basement membrane-like structures containing perlecan, collagen IV and laminin play a major role in liver differentiation by interacting with immature hepatocytes (Am. J. Pa.
th. 142: 199-208 (1993)). Perlecan is a deficiency of glomerular filtration rate such as proteinuria, diabetic glomerular disease, nephrotic syndrome, and Denys-Dras caused by the above-mentioned change in its structure.
h syndrome (Groffen at al., Nephrol. Dial. Transplant 14: 2119-2129
(1999)), mitogenic and angiogenic diseases (Aviezer et al., Cell 79: 1005-1013.
(1994)), inflammation and tissue repair, ocular and skeletal defect syndromes, and microbial pathogenesis due to infiltration, and the like, have been linked to processes and diseases resulting from the above changes in their structure.

Perlecan core proteins are basement membrane proteins nidogen-1, nidogen-2 and fibrin-2, as well as the Alzheimer's-β amyloid protein (Snow et al. Arch. Biochem. Biophys. 320: 84-95 (1995). )) Binding epitope and platelet growth factor. The protein of SEQ ID NO: 296 or a portion thereof is believed to be a human protein isoform of a basement membrane-like protein, preferably a perlecan protein. Thus, the proteins of the present invention play an important role in their interaction with membrane integrity and other proteins, especially nidogen-1 and 2, LN complexes and heparin compounds. In addition, the proteins of the invention are believed to be involved in interactions with cellular receptors such as integrins, cytokine release and proteolysis, regulation of angiogenesis, wound healing, and tumor invasion. Being overexpressed in fetal liver, the proteins of the invention are believed to be involved in hepatocyte differentiation, migration and adhesion due to their embryonic spatial and temporal expression. A preferred polypeptide of the invention is SEQ ID NO:
It is a polypeptide containing the amino acids at positions 141 to 197 of 296. Other preferred polypeptides of the invention are SEQ ID NO: 296 having the biological activity described herein.
Is a fragment of The activity of the proteins of the invention, or portions thereof, can be assayed as a binding assay with other membrane proteins by Hopf et al., Euro. J. Biochem. 259: 917-925 (1
999), and as a protein assay (immunochemistry and ELISA), Rescan et al., Am.
Assays can be performed using any assay known to those of skill in the art, such as J. Path. 142: 199-208 (1993). In one embodiment, the present invention relates to methods and compositions using the protein of the invention or a part thereof as a novel marker protein to selectively identify the stage of embryogenesis, preferably in liver tissue. Used to selectively identify the time of embryogenesis. For example, any technique known to those of skill in the art can be used to detect a protein of the invention or a portion thereof using a specific antibody capable of binding the protein. Such a tissue-specific antibody is used to identify embryogenic cells having a membrane component that is out of control in differentiated tumor cells, or to differentiate various cell types in a tissue cross section by using immunochemistry. it can. For example,
Using specific antibodies that can be detected by fluorescence (FACS, confocal microscopy, etc.) or any method known to those of skill in the art, the protein of the invention can be expressed in embryogenic cells that exhibit characterized overexpression activity. The amount is measured and compared to normal cells. Books in embryogenic cells that show characterized overexpression activity using, for example, specific antibodies that can be detected using fluorescence (FACS, confocal microscopy, etc.) or any method known to those of skill in the art. The amount of protein of the invention is measured and compared to normal cells.

Another embodiment of the invention relates to methods and compositions using the protein of the invention or a part thereof for diagnosing disorders associated with overexpression of the protein of the invention, preferably these Human melanoma, proliferative diseases, lack of glomerular filtration rate such as proteinuria, diabetic glomerulopathy, nephrotic syndrome, De
It has applications in the diagnosis of nys-Drash syndrome, mitogenic and angiogenic diseases, inflammation and tissue repair, ocular and skeletal defect syndrome, and microbial pathogenesis due to infiltration. Any method well known to those skilled in the art, such as Northern blotting, RT-PCR, or immunoblotting using a specific antibody that binds to the protein of the present invention can be used to detect the protein of the present invention. Expression can be examined. In yet another embodiment, the proteins of the invention, or portions thereof, stimulate the growth and differentiation of various cells, including but not limited to fibroblasts, myocytes, osteoblasts, keratinocytes and hepatocytes. Can be used as a mitogen for In a preferred embodiment, the protein of the invention or a portion thereof is used in in vitro culture, such as for synthesizing recombinant proteins. An amount of a protein of the invention or a portion thereof effective to stimulate proliferation and / or differentiation is added to the culture. A more preferred use of this technique is to produce or regenerate the proteins or portions thereof of the invention in vitro for transplantation and transplantation, including but not limited to skin, cartilage and bone, etc., tissues and organs. (Lancet-1981, 1 (8211): 75-8), the disclosure of which is incorporated herein by reference in its entirety. In another embodiment, SEQ ID NO: 296 is for the treatment or prevention of cell proliferative disorders.
Can be administered to a subject to be treated. Such disorders include arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease.
(MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, And especially the adrenal gland, bladder, bone, bone marrow, brain, breast, neck, gallbladder, ganglia,
Digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate,
Includes but is not limited to cancers of the salivary glands, skin, spleen, testes, thymus, thyroid and uterus. In one embodiment, an antibody that specifically binds to a protein of the invention is used to transfer a formulated substance directly to the cell or tissue that expresses the protein of the invention, either as an antagonist or indirectly. Can be used as a targeting or delivery mechanism for In another example, an antisense nucleotide designed from nucleotides encoding a protein of the invention or a portion thereof to inhibit expression of a protein of the invention using methods known to those of skill in the art,
Administer triple helix, Genetic Suppressor Elements (GSE) or ribozyme.

<Protein of SEQ ID NO: 410 (Internal Designation 179-9-4-0-B8-CS)> The protein of SEQ ID NO: 410 encoded by the cDNA of SEQ ID NO: 169 is present in the kidney of a fetus and is an ankyrin family. And a protein having a characteristic ankyrin repeat motif (pfam accession number PF00023). The protein of the present invention is a human ankyrin protein (PIR Accession No. A35049, SP-TR
EMBL accession number Q99407) is an ankyrin (Drosophila melanogaster): STR accession number Q9VAU5, mouse: S from several different eukaryotic species.
TR deposit number Q61302 and SwissProt deposit number Q02357, cattle: STR deposit number AAF617
02, plants (Arabidopsis thaliana): STR Accession No. Q9ZQ79) and homologues of prokaryotes-derived ankyrin (Paramecium bursaria Chlorella virus: STR Accession No. STRQ41164). Furthermore, the proteins of the invention show homology with proteins containing other ankyrin repeat motifs. The ankyrin repeat motif is a 33 amino acid motif with an L-shaped structure consisting of two alpha helices followed by a β hairpin loop (Gorina et al., Science.274-1005 (1996)). As proteins containing ankyrin repeats, channels, enzyme poisons, transcription factors (Palek et al., Semin. Hemato.
l. 27: 290-332 (1990)), tankyrase (Smith et al., Science. 282: 1484-1487 (1
998)), a number of proteins involved in signal transduction, especially integrin-binding kinases (Huang et al., Int. Mol. Med. 3: 563-572 (1999)), inhibitors of cyclin-dependent kinases (Baumgartner et al., Structure. 6 :). 1279-1290 (1998)), death-related protein kinase for apoptosis (Raveh et al., Proc. Nath. Acad. U.
SA. 15: 1572-1577 (2000)) and many others. The ankyrin motif is also present in the proteins of the invention (positions 47-79). Ankyrin is a peripheral membrane protein whose presence was found in mammalian red blood cells, kidney cells and neuronal cells. Cells contain a cytoskeleton, which connects multiple intracellular compartments to each other, and the plasma membrane. The association of the aforementioned cytoskeleton with the lipid membrane that serves as the boundary between the plurality of intracellular compartments includes spectrin, ankyrin and integral membrane proteins. Spectrin is a major component of the cytoskeleton, which acts as a scaffold protein. Similarly, ankyrin acts to connect the actin-spectrin moiety to the membrane and to regulate the interaction of the membrane compartment with the cytoskeleton. Different ankyrin isoforms are specific for different organelles, providing specificity for this interaction. The genes encoding three different mammalian ankyrins (Ankyrin R, Ankyrin B and Ankyrin G) have been cloned. Ankyrin R was previously identified as part of the erythrocyte membrane skeleton and has recently been mapped to the plasma membrane of a subpopulation of postmitotic neurons in the rat brain (Lambert et al.,
1993, J. Neurosci., 13, 3725-3735). Ankyrin B is a developmentally regulated human brain protein that has two alternatively spliced isoforms with molecular weights of 220 kilodaltons (kD) and 440 kD, respectively (K
unimoto et al., 1991, J. Cell Biology, 115, 1319-1331). Ankyrin G is a more recently isolated human gene that encodes two neurospecific ankyrin mutants (480 kD and 270 kD), which are the ankyrin origins of Lambier. And located in the nodule (Kordeli et al., 1995, J. Biol.
Chem., 270, 2352-2359). Studies on mammalian ankyrins show that ankyrins bind to various proteins, which are associated with an anion exchanger (Drenckhah
n et al., 1988, Science, 230, 1287-1289), Na + / K + ATP degrading enzyme in the kidney, amiloride-sensitive Na channel (Smith et al., 1991, Proc. Natl. Acad. Sci.
USA, 88, 6971-6975), voltage-gated Na channels at the brain and neuromuscular junction (S
rinivasan et al., 1988, Nature, 333, 177-180) and neural cell adhesion molecules (
Davis et al., 1994, J. Biol. Chem., 269, 27163-27166).

Analysis of the mammalian ankyrin shows that this large protein is divided into three functional domains. These are membrane-binding domains of the N-terminal of approximately 89-95 kD, approximately
It contains a 62 kD spectrin binding domain and a C-terminal regulatory domain of approximately 50-55 kD. The membrane-binding domain consists mainly of tandem repeats, each having about 33 amino acids. This domain usually contains approximately 22-24 copies of these repeats. This repeat unit is believed to function in binding to membrane proteins such as anion exchangers, Na channels, and certain adhesion molecules. As its name implies, the spectrin binding domain functions to bind to the cytoskeleton, which contains spectrin as the major protein of cells located within the plasma membrane. Ultimately, the regulatory domain is the most mutated domain of the ankyrins studied, but is believed to function as a repressor and / or activator of the protein binding activity of the other two domains. . In different Ankyrin species,
The variability seen in this domain is believed to be due to alternative splicing of nascent transcripts. Regulatory domains can respond to cell signals and enable remodeling of the cytoskeleton during cell cycle and differentiation (Lambert, S. and Bennet
t, V. (1993) Eur. J. Biochem. 211: 1-6). Ankyrin is thought to be the target of parasitic effects. The red blood cell ankyrin is associated with tropical malaria parasites.
(falciparum), which is cleaved by a parasitic proteolytic enzyme, destabilizes the membrane skeleton of erythrocytes and promotes parasite release (Raphael et al., Mol Biochem Parasitol. 11
0 (2): 259-272 (2000)). Recently, novel ankyrin proteins have been isolated from canine filamentous and filamentous worms and are believed to be useful in protecting animals such as humans from diseases caused by parasitic helminths (US Pat. No. 6,063,599). Ankyrin sequences have been identified in diverse libraries, with at least 50% associated with cancer and at least 23% associated with immune response. Of particular note are reproductive and hematopoietic / immune and ANFP expression in gastrointestinal tissues. US Patent 5
, 989,863. Since the protein of SEQ ID NO: 410 belongs to the human ankyrin protein family, it is thought to play a role in controlling the interaction between cytoskeleton and membrane components. The identification of new ankyrin family members and polynucleotides encoding them has led to the identification of diagnosis, prevention and treatment of autoimmune / inflammatory, cell proliferative, and vesicular trafficking disorders, and response to infectious diseases. It is shown to meet the needs in the art by providing new compositions that are useful for modulating.

Preferred polypeptides of the present invention are those containing amino acids at positions 47-79. Other preferred polypeptides are SEQ ID NO: 41 having the desired biological activity.
It is a fragment of 0. In addition, the polypeptide encoded by the human cDNA of clone 179-9-4-0-B8-CS is included in the invention. The polypeptide of SEQ ID NO: 410 can be replaced with the corresponding polypeptide encoded by the human cDNA of clone 179-9-4-0-B8-CS. Further, a polynucleotide encoding the polypeptide is also included in the present invention. A preferred polynucleotide is SEQ ID NO:
169 and the clone 179-9-4-0-B8-CS human cDNA polynucleotides. The present invention also includes variants of the proteins of the present invention. Preferred variants are
At least about 80%, more preferably at least about 90%, and most preferably at least about 95% of the amino acid sequence of SEQ ID NO: 410 is in agreement and has at least one functional or structural ankyrin characteristic. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising the sequence of SEQ ID NO: 410 and variants thereof. Variants encoding features of at least one functional region of the ankyrin proteins of the invention are included. The use of codons may be different, depending on the standard method used, to enhance expression in different hosts. In one embodiment, the protein of SEQ ID NO: 410 or a fragment or derivative thereof can be administered to a therapeutic subject to treat a disorder associated with decreased expression or activity of ankyrin. Examples of such disorders include autoimmune / inflammatory disorders such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atheroma. Atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyglandular endocrine disorders-Candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis , Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, incidental lymphopenia associated with lymphocytotoxic factor, fetal erythroblastosis, erythema nodosum, atrophic gastritis, glomerulonephritis Goodpasture syndrome , Gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis , Polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome , Cancer, hemodialysis and extracorporeal circulation complications, viral, bacterial, fungal, parasitic, protozoal and parasite-like infections and trauma; actinic keratosis, arteries as cell proliferative disorders Sclerosis, bursitis,
Cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, as well as adenocarcinoma, leukemia, lymphoma, melanoma,
Cancers such as myeloma, sarcoma, teratocarcinoma, and especially adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, Cancers of the parathyroid gland, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid and uterus; also impaired vesicular transport, cystic fibrosis, glucose-galactose malabsorption syndrome, hypercholesterolemia, Diabetes, diabetes insipidus, hyper and hypoglycemia, Grave's disease, goiter and Cushing, ulcerative colitis and gastric or duodenal ulcer, but are not limited thereto.

In another embodiment, a vector capable of expressing the protein of SEQ ID NO: 410 or a fragment or derivative thereof for treating disorders related to reduced expression or activity of ankyrin, including but not limited to the disorders described above. Can be administered to a patient. In yet another embodiment, the protein of SEQ ID NO: 410 or a portion of that protein for treating or preventing the disorders described above, including but not limited to, decreased expression or activity of the same or similar proteins. A pharmaceutical composition containing the highly purified protein of 1. with a suitable drug carrier can be administered to a subject to be treated. In yet other embodiments, sequences that modulate the activity of the protein or other ankyrin protein to treat or prevent the disorders described above, including but not limited to, decreased expression or activity of the protein. Number 410
An agonist of the protein can be administered to the patient. In another embodiment, the polypeptide of SEQ ID NO: 410 can be used to generate an antagonist using methods known in the art. In particular, the purified polypeptide can be used for the production of antibodies, or for screening libraries of pharmaceutical agents to identify those that specifically bind to ankyrin proteins. Neutralizing antibodies (ie, those that inhibit dimer formation) can also be prepared for therapeutic use. In another embodiment, an antagonist of the protein of SEQ ID NO: 410 can be administered to a patient to treat or prevent a disorder associated with increased expression or activity of the protein or another protein of the ankyrin protein family. Such disorders may include, but are not limited to, those listed above. In one embodiment, targeting for transfer of an agent to a cell or tissue that expresses the polypeptide, either directly as an antagonist or indirectly by an antibody that specifically binds to the polypeptide claimed in this application. Or it can be used as a delivery mechanism. In yet another embodiment, a vector capable of expressing the complement of the polynucleotide of SEQ ID NO: 169 to treat or prevent disorders associated with increased expression or activity of ankyrin protein, including but not limited to the disorders described above. Can be administered to patients.

In other embodiments, any protein, antagonist, antibody, agonist, complementary sequence or vector of the invention can be administered with a suitable therapeutic agent. Combinations of therapeutic agents may act synergistically to treat or prevent the various disorders described above. Using this method, small doses of each drug could provide therapeutic efficacy and reduce the potential for adverse side effects. In another embodiment of the invention, the polynucleotide encoding the polypeptide of SEQ ID NO: 410, or any fragment or complement thereof, can be used therapeutically. In one embodiment, if it is desired to block the transcription of mRNA, the complement of the polynucleotide encoding the above-mentioned polypeptide can be used. In particular, cells can be transformed with a complementary sequence of a polynucleotide encoding the above-mentioned polypeptide. Thus, complementary molecules or fragments can be used to modulate the activity of the claimed polypeptides or related ankyrin proteins, or to control gene function. In another embodiment of the invention, the nucleotide sequence encoding the polypeptide of SEQ ID NO: 410 can be used to block the gene expressing the polynucleotide or related ankyrin protein. In particular, cells or tissues can be transformed with expression vectors that express high levels of polynucleotides or fragments thereof. Such constructs can be used to introduce untranslated sense or antisense sequences into cells. Expression vectors derived from retroviruses, adenoviruses or herpes or vaccinia viruses or various bacterial plasmids can be used to deliver the nucleotide sequences to target organs, tissues or cell populations. Another embodiment of the invention relates to administering the formulation, or a sterilized composition, in association with a pharmaceutically acceptable carrier to achieve any of the above therapeutic effects. The composition can be delivered by a variety of routes. In another embodiment, the sequence is used for the diagnosis of disorders characterized by the expression of ANFP, or for assays that monitor patients being treated with the polypeptide, other ankyrin proteins or their agonists, antagonists or inhibitors. Number 410
An antibody that binds to the polypeptide of can be used. Various assays can be used, such as ELISAs, RIAs, FACS, etc.

In another embodiment of the invention, the polynucleotide of SEQ ID NO: 169 may itself be used for diagnosis. Oligonucleotide sequences, complementary RNA and DNA useful in diagnostics
The polynucleotides can be used to generate molecules as well as PNAs. The polynucleotides and related molecules can be used to detect and quantify gene expression in tissue undergoing biopsy, where expression of the polypeptide of SEQ ID NO: 410 or other ankyrin polypeptides can be correlated with disease. . Diagnostic assays can be used to determine the absence, presence and overexpression of the polypeptide and to monitor the control of the amount of the polypeptide during therapeutic intervention. Examples of diagnostic methods include Southern or Northern analysis, dot blot or other membrane-based techniques, PCR, dipstick, pin and multi-format ELISA-type assays, and patient fluids or tissues for ANFP analysis. Microassay methods that detect altered expression are included. Such qualitative or quantitative methods are known in the art. Such assays can also be used in clinical trials in animal studies to assess the efficacy of a dosing regimen in a particular treatment, or to monitor treatment in individual patients. In another embodiment, oligonucleotides or longer fragments derived from the polynucleotide sequences described herein can be used as targets in microarray methods. The microassay can be used to simultaneously monitor the expression levels of multiple genes and to identify genetic variants, mutations and polymorphisms.
This information can be used to determine the function of genes, understand the genetic mechanism of disorders, diagnose disorders, and develop and monitor therapeutic agents. In another aspect of the invention, the polypeptides can be used to stimulate the expression of genes that play a role in organ and organ system development. Thus, in a preferred embodiment, the protein of the invention or a fragment or derivative thereof can be administered to a patient to treat or prevent a developmental disorder. In another embodiment, the proteins of the invention can be administered to a patient to treat or prevent cardiovascular disorders. Such disorders include arteriosclerosis including atherosclerosis and non-atherosclerosis, hypertension, stroke, coronary artery disease, ischemia, myocardial infarction, angina, cardiac arrhythmia, sinus node block, atrioventricular node. Examples include, but are not limited to, block, chronic hemodynamic overload, aneurysm, Gerber Lange Nielsen syndrome and long QT syndrome. Since the protein of the present invention can also be used as a marker of cardiac hypertrophy, it can be included in a diagnostic kit for this disease.

In other embodiments of the invention, the polypeptides and / or polynucleotides can be used to inhibit cell proliferation or to treat and / or diagnose cell proliferation related disorders such as cancer. In another embodiment, an antagonist of a protein of the invention can be administered to a patient to treat or prevent cancer. In one embodiment, an antibody that specifically binds to a protein of the invention is used as a target, either directly as an antagonist or indirectly to transfer the dispensed drug to cells or tissues that express the protein of the invention. It can be used as an emulsification or delivery mechanism. In yet another embodiment, an antagonist of a protein of the invention can be administered to a subject to treat or prevent a neuronal disorder. Such disorders include akathisia, Alzheimer's disease, amnesia, amyotrophic lateral sclerosis, bipolar disorder, catatonic disease, cerebral tumor, dementia, suppression, diabetic neuropathy, Down syndrome, delayed movement disorder. , Dystonia, epilepsy, Huntington's disease, peripheral neuropathy, multiple sclerosis, neurofibromatosis, Parkinson's disease, paranoid psychosis, postherpetic neuralgia, schizophrenia and Tourette's disorder. Not limited to. In one embodiment, targeting to transfer a dispensed drug to a cell or tissue expressing a protein of the invention, either directly as an antagonist, or indirectly with an antibody that specifically binds the protein of the invention. Or it can be used as a delivery mechanism. In other embodiments, the proteins of the invention can be administered to a subject to treat or prevent malaria. The protein of the present invention can also be used for the diagnosis of malaria. In yet another embodiment, the polynucleotides and / or polypeptides of the present invention can be used as a therapeutic composition capable of protecting an animal from a disease caused by a parasitic helminth. The polypeptide can be used as a target for anthelmintic vaccines and drugs. Ankyrins have been shown to underlie membrane proteins including CD44, voltage-gated Na channels, NA + / K + ATP-degrading enzymes and anion exchange proteins. Forming a direct link between ankyrin and a functionally important transmembrane protein / membrane scaffold is believed to be one of the earliest events in signal transduction and cell activation. Thus, in another embodiment, the polypeptides of the invention can be used to break the link between ankyrin and the membrane, thus affecting basic processes within the cell.

The polypeptides of the present invention can be used to screen for compounds that inhibit or enhance the binding of ankyrin-binding proteins, or to proteins that are important for intracellular ion and nutrient transport such as α-Na and K-ATP degrading enzymes. Can be used to influence association with ankyrin. In yet another embodiment, the regulatory domain of the polypeptide of SEQ ID NO: 410 or an antagonist thereof is used to inhibit or enhance the protein binding activity of other domains. <Proteins of SEQ ID NOs: 385 and 416 (Internal designation 105-021-3-0-C3-CS, respectively)
And 188-31-1-0-E6-CS)> The protein of SEQ ID NO: 385, which consists of 354 amino acids and is encoded by the cDNA of SEQ ID NO: 144, is present in the brain and is 20-66, 68-114, 116-162. , 164-209, 211-26
Six Kerch motifs (pfam accession number PF01344) are shown at positions 5 and 270-316. In addition, the four conserved residues in 90% or more of the Kerch family sequences are
It exists in the protein of the present invention, diglycine is located at positions 133 to 134, and tyrosine is located at 148.
And tryptophan at 155. In addition, the tyrosine 148 and the tryptophan 155
Are separated by 6 residues and this feature is conserved in over 70% of Kerch proteins
(Adams et al., Trends in cell biology, 10: 17-24, 2000). CDN of SEQ ID NO: 144
The protein of the invention encoded by A is a polymorphic variant of the protein of SEQ ID NO: 416 encoded by the cDNA of SEQ ID NO: 175 and is believed to have similar function and utility. Drosophila querch is located in the actin-rich bridge, the circular tube. Kerch is localized at the rim of the shaped tube and acts to maintain actin tissue (Xue et al., Cell (72) 681-693, 1993; Robinson et al., J. Cell Biol. (138) 799-810. ,
1997). In mammalian sperm, calicin is located in an actin-negative structure called the calyx, which plays a role in spermatocyte morphogenesis (von Bulow et al., Exp. C).
ell Res. (219) 407-413, 1995). In some teratospermia, the absence of calicin and the well-constructed calyx suggests that calicin plays a central role in tissues of this structure (Courtot et al., Mol. Reprod. Dev. (28) 272. -279, 1
991) .In Schizosaccharomyces pombe (fission yeast), Ral2p is downstream of Ras1p,
It acts by pathways that affect cell morphology, mating and sporulation. The globular morphology and junctional defects of ral2 null cells are complemented by Ras1p overexpression, indicating a close functional interaction between both proteins (Fukui et al., Mol. Cell. Biol.
(9) 5617-5622, 1989). Under normal cellular conditions, the transcription factor Nrf2 is sequestered by kerchiripito containing the Keap1 protein. Stimulation with a drug such as diethylmaleic acid induces translocation to the Nrf2 nucleus, resulting in a cytoprotective electrophilic counterattack (Itoh et al., Genes Dev. (13) 76-86, 1999). Lytic infection of cells by herpes simplex virus is initiated by the binding of the virally encoded VP16 to HCF-1, a protein thought to play a normal role in cell cycle progression. The HCF-VP16 complex then aggregates with the Oct-1 transcription factor on cis-regulated targets of the HSV genome and initiates viral replication (Wilson et al. Mol. Cell. Biol (17) 6139.
-6146, 1997; Hughes et al., J. Biol. Chem. (274) 16437-16443, 1999). Two recently discovered mammalian kerchiripito proteins play several roles extracellularly. Human attractin is believed to be involved in normal immune defense as a serum glycoprotein released by activated T cells. In co-culture assays, attractin stimulates monocyte adhesion and propagation, promoting T cell clusters and cellular immune responses (Duke-Cohan et al., Proc. Natl. Acad. Sci. USA).
(95) 11336-11341, 1998). Attractin is a homologous homologue to the extracellular domain of mouse mahogany, which is a large multidomain transmembrane protein that has an inhibitory effect on certain types of obesity in mice. And is associated with homeostasis in humans (Gunn et al., Nature (398).
152-156, 1999; Nagle et al., Nature (398) 148-152, 1999).

The importance of the (-propeller of kerchiripito in protein function has been shown from studies on natural and artificial loss-of-function mutations. Caenorhabditis eleg
Ans Spe-26 mutant spermatocytes have not completed meiosis and have multiple nuclei with extreme tissue disruption in actin filaments and organelles. For 5 of the 6 alleles examined in detail, the mutations are mapped within the kerchirepeat (Varkey et al., Genes Dev. (9) 1074-1086, 1995). Of particular note is a point mutation in RAG-2 identified in several cases of human B-cell negative severe combined immunodeficiency or Omen syndrome (Schwarz et al., Science).
(274) 97-99, 1996; Villa et al., Cell (93) 885-896, 1998). More recently, gigaxonin, a new member of the BTB / kertilite family of cytoskeletons, has been described as mutated in giant axon neuropathy (Bomont et al. Nat.
Genet. (26) 370-374, 2000). The proteins of the invention belong to the superfamily of Kerch and are therefore associated with the actin cytoskeleton, the organization of the cytoskeleton, the structure of the plasma membrane or organelle, the coordination of morphology and growth, gene expression, viral pathogenesis and It is believed to play a role in immune defense. The proteins of the invention are highly expressed, especially in the brain, and are believed to be associated with disorders of the central nervous system. Preferred polypeptides of the present invention include amino acids of the proteins of the present invention 20-66, 68-114, 116-162, 164-209, 211-265.
And at positions 270-316. In one embodiment, the proteins of the invention, or portions thereof, are used to modulate actin organization within cells and can affect cytoskeleton and cell function in general. The invention also features compounds, such as proteins, that interact with the proteins of the invention. Any method suitable for detecting protein-protein interactions can be used to identify transmembrane, intracellular or extracellular proteins that interact with the protein. Traditional methods that can be used include co-immunoprecipitation, cross-linking,
And a method of co-purifying cell lysates or proteins obtained from cell lysates through a gradient or chromatographic column, and methods of using the proteins of the invention to identify proteins in lysates that interact with them. is there.
In these assays, the proteins of the invention may be full length or other suitable protein polypeptide fragments. Once isolated, such interacting proteins can be identified and cloned and then used in a conventional manner to identify interacting proteins. For example, at least a part of the amino acid sequence of the protein that interacts with the protein of the present invention can be determined by a technique known to those skilled in the art, such as Edman degradation method. The amino acid sequence thus obtained can be used as a guide in generating an oligonucleotide mixture for screening gene sequences encoding the interacting proteins described above. The screening can be performed by, for example, standard hybridization method or PCR method. Methods for producing and screening oligonucleotide mixtures are known. ("PCR Protocols: A Guide to Methods and Applica
tions, "Innis et al., eds. Academic Press, Inc., NY, 1990).

Furthermore, a method for directly identifying a gene encoding a protein that interacts with the protein of the present invention can be used. These methods include, for example, a labeled polypeptide, or a protein fusion protein, for example, a marker such as an enzyme, a fluorescent dye, and a luminescent protein, or an IgFc domain, as in the known antibody probe technology of a λgt11 library. , A method of screening an expression library using the fused protein, polypeptide or domain. Another embodiment of the invention is a composition that uses a protein of the invention, or a portion thereof, to modulate actin tissue and associated cytoskeletal protein organization in cells, particularly cells of the central nervous system. And about the method. Compositions comprising the proteins of the invention and fragments thereof are of therapeutic value and can be used to treat various neuronal disorders. Another embodiment of the invention relates to administering the formulated or sterilized composition in association with a pharmaceutically acceptable carrier to achieve any of the above therapeutic effects. The composition can be delivered by a variety of routes. In yet another embodiment, an antagonist of a protein of the invention can be administered to a subject to treat or prevent a neuronal disorder. Such disorders include akathisia, Alzheimer's disease, amnesia, amyotrophic lateral sclerosis, bipolar disorder, catatonic disease, cerebral tumor, dementia, suppression, diabetic neuropathy, Down syndrome, delayed movement disorder. , Dystonia, epilepsy, Huntington's disease, peripheral neuropathy, multiple sclerosis, neurofibromatosis, Parkinson's disease, paranoid psychosis, postherpetic neuralgia, schizophrenia and Tourette's disorder. Not limited to. In one embodiment, an antibody that specifically binds to a protein of the invention is used to transfer the dispensed drug to a cell or tissue that expresses the protein of the invention, either directly as an antagonist or indirectly. It can be used as a targeting or delivery mechanism. Other embodiments of the invention include DNA sequences encoding proteins of the invention that are derived by biological or synthetic chemical processes. CDN of SEQ ID NOs: 144 and 175
Polynucleotide sequences capable of hybridizing to the A sequence are also within the scope of the invention.

Further included in the invention are polypeptides encoded by the human cDNAs of clones 105-021-3-0-C3-CS and 188-31-1-0-E6-CS. SEQ ID NOs: 385 and 4
The 16 polypeptides may be replaced with the corresponding polypeptides encoded by the human cDNAs of clones 105-021-3-0-C3-CS and 188-31-1-0-E6-CS. Further, a polynucleotide encoding the polypeptide is included in the present invention.
Preferred polynucleotides are SEQ ID NOs: 144 and 175 and clone 105-021-3-
Human cDNAs for 0-C3-CS and 188-31-1-0-E6-CS. Another embodiment of the invention is a method of using a nucleotide sequence of the invention or a portion thereof to determine a homologue of a protein of the invention. The sequence can be used as a template in a PCR reaction, allowing detection / quantification of the protein of the invention or a part or homologue thereof. The complementary sequence or a part thereof can be used at the in vitro level as a hybridization probe for detecting / quantifying transcription levels as well as at the cellular level. Such probes can be used in particular for diagnostics, for example for on-section hybridisation methods in cells or tissues. Another embodiment of the invention is a nucleotide sequence or part thereof for designing an antisense oligonucleotide for modulating gene expression, which is the expression of a protein of the invention or part thereof in vitro or in vivo. To use. In particular, when the protein of the present invention is expressed at an abnormally high level, it can be useful in the field of treating the above diseases. In another embodiment of the invention, the proteins of the invention or portions thereof are used to raise specific antibodies. These antibodies can be used for diagnostic analysis and purification of the protein of the present invention or a part thereof or a homologue thereof. These antibodies will also be useful in visualizing the location of proteins related to the proteins of the invention, in particular highlighting structurally related proteins. The methods described herein that utilize protein antibodies can be performed using a prepackaged diagnostic kit that includes at least one specific cDNA of SEQ ID NO: or an antibody reagent described herein. For example, it can be conveniently used for diagnosing patients showing various symptoms of central nervous system disorders in medical facilities. In yet another preferred embodiment, the invention provides that the protein of the invention, or a part thereof, is expressed at an abnormally low level, in particular in gene therapy, especially in diseases associated with the central nervous system. Regarding the method to use when. Gene therapy is a potential therapy in which the cDNAs of SEQ ID NOs: 144 and 175
Is a method of efficiently encoding a normal protein in several different affected cell types by introducing a normal copy of P.

Another aspect of the invention comprises a formulation comprising a protein of the invention and a pharmaceutically or physiologically acceptable carrier. The formulations of the present invention include a combination of one or more peptides described herein or a mimetope thereof, or an antibody described herein or a combination of mimetopes thereof, or an antibody described herein. And peptides or their mimetopes. Such formulations can be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of the protein. The disorders described above include, but are not limited to, disorders of the central nervous system and other tissues where actin association is believed to be abnormal. <Proteins of SEQ ID NOs: 391, 393, 405, and 407 (internal designation: 145-52-2, respectively)
-0-D12-CS, 145-7-3-0-D3-CS, 174-17-1-0-D6-CS and 174-38-4-0-D11-CS)> SEQ ID NOS: 150, 152, A cluster of four proteins encoded by 164 and 166, respectively (SEQ ID NOs: 391, 393, 405 and 407) shows high homology to clodine-8, a member of the PMP22 claudine family (PF00822). SEQ ID NO: 405 (174-17-1-0-D6-CS) has a high degree of identity to human claudin-8. SEQ ID NO: 393 has two amino acid substitutions (T129A and S151P) relative to human claudin-8 and is therefore considered a polymorphic variant of claudine-8. SEQ ID NOs: 393 and 405 contain four transmembrane segments. SEQ ID NOs: 391 and 407 are polymorphic variants of claudine-8. SEQ ID NO: 39
The protein of 1 (145-52-2-0-D12-CS) is 162 amino acids long, has 3 theoretical transmembrane segments, and has 3 amino acids compared to the above Claudine-8 protein. Substitutions (R31I, S151P and E162) are shown. The protein of SEQ ID NO: 407 (174-38-4-0-D11-CS) is 43 amino acids long. SEQ ID NO: 407 has a stop codon at position 44 and no putative transmembrane segment. The Claudine protein family consists of over 20 small glycoproteins with four predicted transmembrane domains. The distribution pattern of claudines varies considerably among claudines. Many claudines have been identified as elements of the tight junction (TJ) strand that contribute to cell polarity and permeability. Polarized epithelial and endothelial cells divide the biological compartment and form the barrier that controls homeostasis. Tight junctions (TJs) are specialized membrane domains located in the most apical regions of polar epithelium and endothelial cells that form the major barriers that prevent paracellular transport of solutes (barrier function) and Limits lateral diffusion of membrane lipids and proteins to maintain polarity (fence function). Tight junctions appear to form a continuous network of interconnecting rows of intramembrane particles that appear as strands with complementary grooves. The tight junction-specific integral membrane proteins, the elements of the tight junction strands, occludin and claudine, have been recently identified.

Claudine-1 and claudine-2, when introduced into fibroblasts without tight junctions (TJ), can induce strand / groove network formation at sites of cell-cell contact. Occludin is an accessory protein in the formation of tight junction strands because it induces only a few short strands at the site of cell-cell contact in fibroblasts. Experiments with claudine transfection in fibroblasts have shown that they can form tight junction strands in the absence of occludin (Saitou M et al.
J. Cell Biol. 141: 397-408 (1998), Furuse M et al., J. Cell Biol.
.143: 391-401 (1998)). First, several members of the claudine family (RVP1, Clostridium perfringens enterotoxin receptor (CPE-R) and TMVCF (chromosome 22q11 deletion syndrome <Velo-ca
rdio-facial syndrome>) was reported, but their physiological functions were not determined. Claudine-1 and claudine-2 were identified as elements of a new tight-bond strand (Furuse, M. et al., J. Ce.
II Biol. 141, 1539-1550 (1998)), and showed that CPE-R removes specific claudines from tight junctions. In its presence, the tight junction strands of C3L cells are gradually degraded, significantly reducing the number of tight junction strands and their network complexity (Sonoda N et al., J Cell Biol 147 (1): 195-20).
4 (1999)). In the renal distal tubules, claudine-4 (CPE-R) and claudine-8 colocalize with occludin at their binding complex regions. In the liver,
Claudine-3 and occludin coexist in the bile canaliculi, and tight junction strands are highly and specifically labeled with anti-claudine-3 (Morita et al., PNAS).
96 (2): 511-516 (1999)). Claudines have been shown to form a paracellular diffusion barrier and, surprisingly, are thought to confer channel-like selectivity for solute tissue barrier permeation (Anderson JM and Christina M. .
Van Itallie CM, Current Biology 9: R922-R924 (1999)). In addition to the presence of claudine's multigene family, similar distributions of tight junctions can be inferred from the tissue distribution patterns of each claudine species, contributing to the generation of functional diversity of tight junctions in vivo. . In a single epithelial cell,
Co-expresses two or more different claudines. Claudines interact heterogeneously between each strand of a strand pair, and different claudines ((
It is incorporated together into individual tight junction strands (except in some combinations) (Furuse M et al., J Cell Biol 147 (4): 891-903 (1999)).

Some claudines have been shown to be expression markers for malignant cells. For example, SEMP1 (aging related epithelial membrane protein) is found in adult and fetal liver,
SEMP1 is expressed in healthy human tissues such as pancreas, placenta, renal cortex, prostate and ovary
Is expressed at low or undetectable levels in some breast cancer cell lines (S
wisshelm K et al., Gene 226: 285-295 (1999)). Another claudine family member has been shown to be expressed only in MCF-7 ADR breast cancer cells.
MCF-7 ADR carcinoma cells are estradiol-independent for proliferation, estrogen receptor negative, tamoxifen resistant, vimentin positive, and invasive both in vitro and in vivo (Schiemann S et al. Anticancer Res 17 (1A): 13. -20
(1997)). Down-regulation of claudine-1 expression has also been associated with carcinogenesis in rat salivary gland epithelium (Li D and Mrsny RJ J Cell Biol 148 (4): 791-80.
0 (2000)). The increased microvascular permeability of human glioma, which causes the clinically severe symptoms of cerebral edema, is the result of dysregulation of junctional proteins. Increased tight junction permeability in the colon epithelium, and consequently decreased epithelial barrier function, precedes the development of colon tumors, including carcinomas and adenomatous polyps (Soler AP et al., Carcinogenesis 20 (8):
1425-1431 (1999)). Human Endoendothelial Junctions in Glioblastoma Multiforme Tumor Microvessels Are Depleted of Claudine-1 Expression in Most Tumor Microvessels, but Clodine-5 Is Significantly Down in Hyperplastic Ducts Controlled. The association between claudine-1 repression and tight junction morphology changes may correlate with increased endothelial permeability (Liebn
er S et al., Acta Neuropathol (Berl) 100 (3): 323-331 (2000)). The human phenotype of the claudine-16 mutations suggests that it forms channels that diffuse magnesium through the tight junctions of the kidney. Similarly, Claudine-1
1 knockout mice show a role in tight junction formation between Sertoli cells in myelin and testis (Mitic LL et al., Am J Physiol Gastrointest
Liver Physiol 279 (2): G250-254 (2000)). It is believed that the opening of tight junctions by environmental proteinases is an early stage of asthma development for various allergens. The lung epithelium constitutes the barrier that the allergen must pass through before it causes sensitization. Dermatophagoides pt
D from the stool mass of eronyssinus (House Dust Mite, House Dust Mite (HDM))
The er p 1 cysteine proteolytic enzyme allergen disrupts tight junctions and tight junctions between cells, which are the major components of the epithelial paracellular permeability barrier. Tight junction degradation nonspecifically increases epithelial permeability, allowing Der p 1 to cross the epithelial barrier. A putative Der p 1 cleavage site was found in peptides derived from the extracellular domain of claudine-1. Allergens of the green mosquito mite (HDM) are an important cause of the increased prevalence of asthma (Wan H et al., J Clin Invest 104 (1): 123-3).
3 (1999)).

In many intestinal and systemic diseases, damage to the intestinal barrier is associated with changes in intestinal permeability, which in turn is associated with changes in tight junction function (Gasbarrini G, Montalto.
MItal J Gastroenterol Hepatol 31 (6): 481-488 (1999)). The permeability of tight junctions can be altered by bacterial toxins, cytokines, hormones and drugs.
An oligodendrocyte-specific protein (oligode) present in central nervous system myelin
The ndrocyte-specific protein (OSP) / Claudine-11) is a promising candidate for the involvement of self-antigens in autoimmune demyelinating diseases. In relapsing-remitting multiple sclerosis (MS), anti-OSP antibodies were reported to be present in cerebrospinal fluid. The mouse OSP peptide causes clinical experimental autoimmune encephalomyelitis in animal models of MS and also induces mononuclear cell infiltration and regional demyelination. The OSP peptide also elicits a strong proliferative response in T cells (Stevens DB et al., J Immunol 162: 7501-7509 (1999).
)). OSP / Claudine-11 appears to modulate oligodendrocyte proliferation and migration, believed to be due to membrane interactions with extracellular matrix in tight junctions (Bronstein JM et al., J Neurosci Res 59). (6): 706-711 (2000))
. Recently, claudine-11 has been shown to play an important role in the formation of the blood-testis barrier, a process that is regulated by FS hormones and cytokines in the early womb and in postnatal development of Sertoli cells. (Hellani
A. et al., Endocrinology 141: 3012-3019 (2000)). SEQ ID NOs: 391, 393, 405 and 407 are new human proteins with biological activity described for claudine. Nucleic acids encoding the target protein are abundant in the fetal kidney and salivary glands. The invention provides polynucleotides encoding the proteins of SEQ ID NOs: 391, 393, 405 and 407. In one embodiment, the polypeptides of SEQ ID NOs: 391, 393, 405 and 407 are clones 145-52-2-0.
It replaces the polypeptide encoded by -D12-CS, 145-7-3-0-D3-CS, 174-17-1-0-D6-CS and 174-38-4-0-D11-CS. Also provided is the use of this protein or fragments or derivatives thereof (and related polynucleotides) for the diagnosis, treatment and prevention of tumors, and other diseases including disorders associated with altered epithelial function. The variant, as long as it has at least one functional or structural characteristic of a claudine-like protein, is at least about 80%, more preferably at least about 90%, and most preferably at least about 90% of the target protein.
It includes all variants of the protein of interest having 95% amino acid sequence identity.

In one embodiment of the invention, a protein of interest or a bioactive fragment or variant thereof can be administered to a patient to treat or prevent disorders of the salivary glands, kidneys and prostate. The present invention also provides therapeutic regimens for treating epithelial dysfunction and cancer. Disorders that can be treated according to the present invention include asthma, eczema, atopic dermatitis,
Contact dermatitis, static, dermatitis, dermatitis, psoriasis, lichen planus, pityriasis rosea, acne vulgaris, red nose, pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, pemphigoid , Gestational herpes, herpes dermatitis, linear IgA disease, acquired epidermolysis bullosa, dermatomyositis, lupus erythematosus, scleroderma and localized scleroderma; gastritis, peptic ulcer, cholelithiasis, cholecystitis, pancreatitis, Cirrhosis, ulcerative colitis, Crohn's disease and irritable bowel syndrome; Addison's disease, Law syndrome, glomerulonephritis, chronic glomerulonephritis, renal tubular interstitial nephritis, congenital X-linked renal diabetes insipidus, autosomal chromosomes. These include, but are not limited to, dominant, polycystic kidney disease, autoimmune demyelinating disease, multiple sclerosis, glioma and other tumors. Another aspect of the invention provides a method of treating these and / or other pathological conditions by administering to the patient a therapeutically effective amount of one or more proteins of interest. The protein of interest can be administered concurrently or sequentially with a cytokine and / or interleukin that exhibits improved claudine expression. In other embodiments, vectors capable of driving the expression of one or more proteins of interest or biologically active fragments or variants thereof to treat or prevent epithelial permeability disorders, including but not limited to the disorders described above. Can be administered to a patient. Other embodiments of the invention provide compositions and methods for treating or reducing the incidence of asthma, which comprises administering a therapeutically effective amount of a protein of the invention. In one embodiment, purified fragments derived from the extracellular domain of the protein of interest or synthetically engineered peptides can be administered in therapy. Peptides containing putative cleavage sites for environmental allergen proteolytic enzymes can be administered in amounts that competitively inhibit allergen proteolytic enzyme activity. The peptide is
In some cases, it may be designed to bind irreversibly to the allergen and suppress proteolytic enzyme activity.

The adverse effects of normal preservation solutions on epithelial and endothelial permeability in organs to be transplanted are well known (Trocha SD et al. Ann. Surg. 230: 105-113 (1999)).
Increased permeability causes tissue injury and edema. Tissue disruption of tight junction proteins is responsible for the observed tissue injury and edema. Therefore, in other embodiments, the purified protein of interest or variant and / or bioactive fragment thereof can be added to the organ preservation solution to maintain the content and integrity of tight junctions in the organ. In another embodiment, the invention provides a method of producing a "bioartificial" epithelium from non-epithelial cells. The "bioartificial" epithelium produced according to the present invention can be used for reconstructive surgery, epithelial reduction (due to genetic, trauma or tumor) or other therapeutic purposes (eg, burn treatment). “Bioartificial” epithelial cells are obtained by transfecting and reshaping autologous cells of a non-disordered patient as described above. The use of autologous cells to prepare the "bioartificial" epithelial cells of the invention in the treatment of disorders, conditions or diseases associated with epithelial cell loss reduces the risk of tissue rejection commonly found in transplantation methods. , Or remove. Bioartificial tissue engineering methods are well known to those of skill in the art (Machens HG et al., Cells Tissues Organs 167:
88-94 (2000)). In another embodiment, the invention provides an antibody that specifically binds to the proteins of SEQ ID NOs: 391, 393, 405 and 407. The antibodies are SEQ ID NOs: 391, 393, 405 and 407.
It may bind specifically to fragments or variants of the proteins described in. The antibodies of the present invention can also be used to detect proteins of interest in human body fluids, cell extracts or tissue extracts. This assay can be used to diagnose prognosis and disorders of epithelial cancer. This assay can also be used to monitor patients treated with a protein of interest. In other embodiments, polynucleotide sequences encoding proteins of interest or fragments of the polynucleotide sequences can be used to identify or diagnose diseases associated with expression of SEQ ID NOs: 391, 393, 405 and 407. Hybridization assays that can detect the polynucleotide sequences of the invention are well known to those of skill in the art. These assays include, but are not limited to, Northern blots, Southern blots and PCR methods.

Other embodiments of the invention are directed to proteins of SEQ ID NOs: 391, 393, 405 and 407, or variants, immunogenic fragments or proteins thereof, for screening compound libraries in various drug screening techniques. A bioactive fragment is provided. SEQ ID NOs: 391, 393, 405 used for this screening
And 407 proteins, or variants, immunogenic or bioactive fragments of the proteins, released in solution, attached to a solid support, recombinantly expressed on the cell surface, or chemically expressed on the cell surface. Or may be located between cells. The formation of binding complexes between the protein of interest and the agent to be tested can be measured by methods well known to those skilled in the art. Yet another embodiment of the invention provides a method of screening for compounds that modulate epithelial permeability. The polynucleotides encoding the proteins of SEQ ID NOs: 391, 393, 405 and 407, or variants, immunogenic or bioactive fragments thereof, are recombinantly expressed in cells normally lacking tight junctions by the methods described above. can do. Therefore, using this cell, (
Therapeutic modulators can be evaluated for their ability to increase or decrease epithelial cell permeability (eg, based on CPE-like compounds). The compounds identified in this modulator screening method can then be used in therapeutic protocols to modulate epithelial cell permeability, as desired by the physician. The intestinal epithelium is an important barrier to the absorption of aqueous drugs. The presence of intercellular junction complexes, particularly tight junctions, renders the epithelium impermeable to hydrophilic drugs that cannot diffuse between cells through lipid bilayers (Ward PD et al., Pharmaceutical Science an
d Technology Today 3: 10: 346-358 (2000)). Therefore, in another embodiment of the invention, the protein of SEQ ID NO: A, B, C or D, or a variant of said protein, or a biologically active fragment of said protein and its molecular partner is It can be used for rational design of compounds that can increase paracellular permeability efficiently and safely. For example, a polynucleotide encoding the protein of interest, or a fragment or derivative thereof can be used for this purpose. In one embodiment, particularly for temporarily increasing epithelial permeability (useful for drug delivery),
If it is desired to block the transcription of the mRNA encoding the protein of interest, then the complement of the polynucleotide encoding the polypeptide can be used. Alternatively, a polynucleotide sequence in which the sense or antisense oligonucleotides encode proteins of SEQ ID NOs: 391, 393, 405 and 407, or variants of the protein, or biologically active fragments to control expression of the protein. It can be designed from various positions in the code or control area. Methods of making and using sense or antisense oligonucleotides are well known to those of skill in the art.

Claudine is a unique protein with specific protein binding characteristics. Therefore, in other preferred embodiments, SEQ ID NOs: 391, 393, 405 and 407.
Or a variant or biologically active fragment of the protein of
It can be used as an element of drug delivery vehicles such as colloids or liposomes. Sequence number
The proteins of 391, 393, 405 and 407, or mutants or bioactive fragments of the proteins, are taken up by the lipid membrane of liposomes and bind to specific epithelial targets as specific targeting agents for epithelial targeted drug delivery. To facilitate. Methods of designing this drug delivery system are well known to those skilled in the art (Smith HJ Introduc
tion to the principles of drug design and action, 3rd ed. (19
98)). Alternatively, the proteins of SEQ ID NOs: 391, 393, 405 and 407, or variants of the proteins, or bioactive fragments are chemically or recombinantly combined with an active agent such as a chemotherapeutic agent, radioisotope, prodrug, Can be attached directly and used in therapy. <Proteins of SEQ ID NOs: 278, 282 and 300 (internal designation: 160-37-2-0-H7
-CS, 174-33-3-0-F6-CS and 184-4-1-0-A11-CS)> SEQ ID NO: 278 (SEQ ID NO: 278 encoded by the cDNA of SEQ ID NO: 37 (41 and 59, respectively))
And the proteins of the corresponding allelic variants 282 and 300) show homology with the human transmembrane protein (HTMN-23, Genseq accession number Y57899). SEQ ID NO: 278 (and the corresponding polymorphic variant 282 and
300) proteins are 85-105, 116-136, 164-184, 187-207, 332-352, 37.
It contains cryptic transmembrane segments at nine positions 6-396, 404-424, 465-485 and 499-519, thus exhibiting the characteristic properties of transmembrane protein type III (Singer, Annu.
Rev. Cell Biol., 6: 247-296, 1990). In addition, the protein of the present invention is
R) is predicted to be localized, but this localization has been confirmed by psort software. The proper functioning of eukaryotic cells requires that all newly synthesized proteins be correctly folded, modified and delivered to specific intracellular and extracellular sites. Newly synthesized membrane and secreted proteins enter the cell's sorting and distribution network during or shortly after synthesis and are routed to specific intracellular or extracellular locations. The first compartment in this process is the endoplasmic reticulum (ER)
However, here, the protein undergoes modifications such as glycosylation, disulfide bond formation and oligomer constitution. The modified protein is then transported through a series of membrane-bound compartments, including the diverse Cisterna of the Golgi complex, for further carbohydrate modification. Budding and fusion occurs between compartments by vesicles that are specific to the type of protein transported. Once the protein enters the secretory pathway, it does not need to cross the membrane to reach the cell surface. Disruption of cells in the secretory pathway has been associated with several human diseases. In familial hypercholesterolemia, low density lipoprotein receptors remain in the ER rather than translocate to the cell surface (Pathak et al., J.
Cell Biol., 106: 1831-1841, 1988). β-amyloid precursor protein (betaAP
Altered transport and processing of P) involves the putative vesicle transport protein prenesilin and may contribute to early-onset Alzheimer's disease (Levy-Lahad et al., Science, 269 :). 973-977, 1995). Changes in ER-derived calcium homeostasis include cardiomyopathy, cardiac hypertrophy, myotonic dystrophy, Brody's disease, Smith-M
It is associated with cCort dysplasia and diabetes mellitus.

The protein of the present invention is believed to be a new ER endogenous transmembrane protein. This protein probably contributes to the post-translational modification of secretory and membrane proteins. The onset of its deregulation may be associated with disorders such as the diseases mentioned above. Preferred polypeptides of the present invention are 85-105, 116-136, 1
64-184, 187-207, 332-352, 376-396, 404-424, 465-485 and 499-519
Is a polypeptide comprising amino acids of SEQ ID NOs: 278, 282 and 300 from the position Other preferred polypeptides of the present invention are fragments of SEQ ID NOs: 278, 282 and 300 that have any of the biological activities described herein. One object of the invention is compositions and methods for targeting a heterologous polypeptide to the endoplasmic reticulum by recombinantly or chemically fusing a fragment of the protein of the invention to the heterologous polypeptide. A preferred fragment is a protein of the invention or a portion thereof that contains a targeting signal to the endoplasmic reticulum, for which Pidoux AL, Armstrong EMBO J 1992 Apr; 11 (4): 1583-9.
1; Munro S, Pelham HR Cell 1987 Mar 13; 48 (5): 899-907; Pelham HR Tren
ds Biochem Sci 1990 Dec; 15 (12): 483-6. In another embodiment, the invention provides a protein of the invention, or a portion thereof, to tissue,
Preferably it relates to methods and compositions using saliva as a marker protein for selective identification. For example, the proteins of the invention, or portions thereof, can be used to synthesize specific antibodies using techniques known to those of skill in the art, including those described herein. Such tissue-specific antibodies can then identify tissues of unknown origin, such as differentiated tumor tissues that have metastasized to forensic samples, different body sites, etc., or use immunochemistry to distinguish different tissue types in tissue sections. Can be used for In addition, antibodies to the proteins of the invention, or portions thereof, can be used to detect endoplasmic reticulum immunochemically using techniques known to those of skill in the art, including, for example, those described herein. <Protein of SEQ ID NO: 281 (174-10-2-F8-CS)> The protein of SEQ ID NO: 281 is homologous to PET117 (SwissProt ID: Q02771). MTC
Is overexpressed in the brain, muscles of muscular dystrophy, fetal liver, placenta and salivary glands.

The protein of the present invention, referred to herein as MTC, has some homology to the yeast PET117 protein precursor material (22% identical amino acids, 39% positive when aligned with BLASTP 2.0.9). Amino acid). MTC is considered a new member of the PET family. Cytochrome C oxidase (complex IV) is an enzyme complex located in the inner mitochondrial membrane and a terminal element of the mitochondrial electron transport system. The oxidation reaction catalyzed by cytochrome C oxidase is energetically linked to the transmembrane transfer of protons. This reaction is a mitochondrial oxidative phosphorylation system that provides the energy needed to drive ATP synthesis and is essential for respiratory metabolism in aerobic eukaryotes. Cytochrome C oxidase consists of as many as 13 different protein subunits, three of which are encoded by the mitochondrial genome and have multiple prosthetic groups (including heme groups a and a3). The organization of cytochrome C oxidase requires multiple discrete steps for the synthesis and organization of an enzyme complex that can act, which is 1) synthesis of protein subunits, 2) from the site of synthesis to the inner mitochondrial membrane. Transport of subunits to the position of action of, 3) synthesis of hemes a and a3, and 4) configuring subunits to each other and to the prosthetic group. Several "accessory" genes (ie, genes that do not encode the protein subunits of the finally assembled cytochrome C oxidase complex) are required to produce functional cytochrome C oxidase (Mc
Ewen JE et al., J Biol Chem, 1986, 261 (25): 11872-9). Some of these are required for expression of the mitochondrial-encoded cytochrome C oxidase subunit, while others are required for the precise organization of active cytochrome C oxidase. The nuclear genes PET117 and PET191 belong to the class of "accessory genes" required for the organization of this active mitochondrial cytochrome C oxidase (McEwen JE et al.
Curr Genet., 1993, 23 (1): 9-14). Although the role of the PET gene and the protein encoded by it is still unknown, mutation experiments in S. cerevisae show that it is essential for the organization of active cytochrome C oxidase (McEwen JE et al., JB.
iol Chem, 1986, 261 (25): 11872-9) (McEwen JE et al., Curr Genet., 1993, 23 (1):
9-14).

One aspect of the present invention provides compositions and methods using the nucleotide sequence of SEQ ID NO: 40, or the complement thereof, in the art of molecular biology. In one embodiment, the MTC2 sequence is encoded by clone 174-10-2-0-F8-CS. SEQ ID NO: 40
To the polynucleotide of SEQ ID NO: 281 and the corresponding polynucleotide of the human cDNA of clone 174-10-2-0-F8-CS and the polypeptide encoded thereby. As the technology, PCR, recombinant MT
C or the production of biologically active fragments thereof, antisense RNA and DNA,
These include, but are not limited to, generation of such chemical analogs, hybridization probes, and chromosomal gene mapping. As will be apparent to those of skill in the art, the techniques described above are not limited to the mt
It can be performed with a fragment of the c2 gene. As the technique is well known, one of skill in the art can select an appropriate length of mtc2 polynucleotide for use in this technique. For recombinant expression of proteins, a preferred embodiment is a full-length expression vector.
Provides the MTC2 gene. For example, the nucleotide sequence of SEQ ID NO: 40, or its complement, can be used to generate hybridization probes for mapping the natural genomic sequence. The sequence can be mapped to a particular chromosome, or to a particular region of a chromosome, using known techniques. This technology is described in Price (Price CM-Blood Rev.
-1993, 7 (2): 127-34) and Trask B (Trask BJ-Trends Genet.-1991, 7 (5).
: 149 54), chromosome spreads, flow-sorted chromosome preparations, artificial chromosome constructs such as yeast artificial chromosomes, bacterial artificial chromosomes, bacterial P1 constructs, or single chromosome Includes in situ hybridization methods for cDNA libraries. Physical mapping, such as on-section hybridization of chromosomal preparations and linkage analysis using established chromosomal markers, is used by researchers searching for pathogenic genes using positional cloning or other gene discovery techniques. It is invaluable for developing a genetic map that provides valuable information. Once a disease or syndrome is localized in a particular genomic region by genetic linkage,
Any sequence that maps to the region can indicate a relevant or regulatory gene to be further studied. The nucleotide sequences of the present invention can also be used to detect differences in chromosomal location due to translocation, inversion, etc. in healthy, carrier or affected individuals.

The present invention also provides for preventing or reducing the incidence of cell apoptosis.
Methods of using MTC polypeptides and polynucleotides encoding the polypeptides are provided. Dysfunction in the mitochondrial electron transport chain causes cell apoptosis or necrosis. In one embodiment, an amount of MTC effective in reducing apoptosis is added to an in vitro culture of mammalian cells. In other embodiments, cells are transfected with a vector containing an MTC polynucleotide to express the MTC peptide. The MTC used in this embodiment may optionally include a mitochondrial targeting sequence. In another embodiment, MTC or MTC2 is encoded over clone 174-10-2-0-F8-CS. In other embodiments, MTC polypeptides and polypeptides encoding the polynucleotides can be used in the diagnosis, treatment and / or prevention of disorders associated with mitochondrial respiratory electron transport, or disorders associated with apoptosis. In certain disorders, the polynucleotides can be used in antisense methods to suppress the action of the mitochondrial electron transport chain. These disorders include immunodeficiency syndrome (AI
(Including DS), Type I diabetes, pathogenic infections, cardiovascular and nerve damage, alopecia, aging, Alzheimer's disease, Parkinson's disease, Huntington's chorea, myotonia, label inherited visual neuropathy , Schizophrenia, neonatal liver failure and necrosis of ketoacidosis coma, as well as "mitochondrial encephalopathy, lactic acidosis and stroke" (MELAS) and "myoclonic tencan clonic myospastic crude red fiber syndrome" (MERRF) Muscle degeneration disorders such as mitochondrial cell degeneration, L
Examples include, but are not limited to, eigh syndrome, fatal infantile cardioencephalopathy, ataxia, encephalopathy, aging, neurodegeneration, myopathy and cancer. As will be apparent to one of skill in the art, this method can be practiced with full length MTC polypeptides and polynucleotides encoding the polypeptides, as well as biologically active fragments thereof that retain biological activity. For diagnostic purposes, expression of the proteins of the invention can be examined using any method known to those of skill in the art, such as Northern blotting, RT-PCR or immunoblotting. For prophylactic and / or therapeutic purposes, SEQ ID NO: 40, its complement or any fragment thereof, enhances electron transfer and energy transfer using any gene therapy known to those of skill in the art. Can be used to Similarly, SEQ ID NO: MTC2, its complement or any fragment thereof, is:
Any antisense method known to any person skilled in the art can be used to suppress electron transfer and reduce energy transfer.

<Protein of SEQ ID NO: 392 (145-7-2-0-G5-CS)> The protein of SEQ ID NO: 392 encoded by the cDNA of SEQ ID NO: 151 is STXBP.
Or it is homologous to Unc-18 protein which is also called Sec-1 family. The protein of the present invention is strongly expressed in fetal kidney. Amino acids 89 to 107 of the protein of the present invention are EM of Sec-1 family proteins.
The otif sign (BlocksPlus PF00995) is shown. Furthermore, the amino acid sequence of the present invention BLAS
Analysis by T (BLASTP Ver. 2.0.9) reveals homology with several proteins of the Unc-18 / Sec-1 family. Preferred polypeptides of the invention include those containing amino acids 94, 95 and / or 100 that are conserved in more than 80% of the Sec-1 family members, and / or amino acids conserved in more than 60% of the Sec-1 family members. It includes 43, 89 and / or 97. Other preferred polypeptides of the invention are any of the fragments of SEQ ID NO: 392, which have any of the biological activities described herein. The normal function and organization of eukaryotic cells is dependent on transport in diverse vesicles that selectively shuttle membranes and cargo between individual compartments of the secretory and endocytic pathways. Several important proteins related to membrane targeting and exocytosis have been identified, revealing a series of basic interactions, SNARE (Soluble N-ethylmaleimi-sensitive adhesion protein receptor;
It has been adopted in a model called the de-sensitive attachment protein REceptor hypothesis (Rotheman J-Nature -1994, 372: p55-63). According to the SNARE hypothesis, vesicles dock to the target membrane by an interaction between complementary vesicle (v-SNARE) and target (t-SNARE) membrane proteins. Characterizing synaptic vesicles in neurons has greatly facilitated our understanding of vesicular transport. In synaptic vesicle exocytosis, vesicle protein synaptobrevin (Vesicle-Associated Membrane Protein; VAMP) is v-SNARE and plasma membrane-associated protein SNAP-25 (25 kDa synaptosome-related protein)
And syntaxin 1 functions as t-SNARE. Α, a cytosolic protein required for membrane fusion after forming the SNARE complex (ie, core complex)
, Β and γ SNAP (Soluble N-ethylmaleimide-sensitive adhesion protein; Solubl
e N-ethylmaleimide-sensitive Attachment Protein) and NSF (N-ethylmaleimide-Sensitive Factor) are supplemented.The proteins from two gene families are important regulators in the formation of SNARE complex. Have been identified as a low molecular weight GTP for these families
There are binding families (eg Rabs) and Sec-1 families. The Sec-1 gene is
It is one of the 10 genes essential for the final stage of protein secretion in yeast (S. cerevisae). Sec-1 homologues have been identified in the nervous system of nematodes (Unc-18), Drosophila melanogaster (Rop) and mammals. In mammals, the protein is the Unc-18 gene (M
unc-18), rbSec1 (rat brain Sec1) or n-Sec1 (neuron-specific Sec1) mammalian homologues.

Sec-1 related proteins are involved in the process of vesicle targeted docking and / or fusion. It has been shown that Sec-1-related proteins interact directly with t-SNARE syntaxin and Munc-18 interacts with syntaxin isoforms 1a, 2 and 3. However, Munc-18 has not been shown to be part of the 20S SNARE / SNAP / NSF protein complex. The binding of Munc-18 to syntaxin vitro showed that syntaxin and VAMP and SNAP-25 and SNAP-23 (SN
AP-25 (homolog), which negatively regulates the synaptic SNARE fusion complex. Consistent with the negative regulatory role of the Sec-1 / Munc-18 protein, the protein in neurotransmitter release was induced by microinjection of Sec-1 into the presynaptic terminals of the synaptic dioica. It is shown that the release of transmitter is suppressed. (Dresbach T. et al.,-J Neurosci.-1998, 1
8: p2923-2932). Furthermore, overexpression of Rop, Unc-18, Sec-1 and Munc-18 all result in a phenotype associated with a complete blockade of neurotransmitter release and / or secretion (Hosono R. Et al-J Neurochem-1992; 58: p1517.
-1525; Harrison S. et al.,-Neuron-1994; 13: p555-566; Novick P. et al.,-Ce.
ll-1981; 25: p461-469; verhage M. et al.,-Science-2000; 287: p864-869).
. Point mutation experiments involving the Rop gene suggest that Rop is a rate-limiting regulator of exocytosis that both stimulates and suppresses neurotransmission (Wu M. et al.
-EMBO J-1998; 17: p127-139). Overexpression of Munc-18 and reduced neurotransmitter release, which is also seen with mutations in the Munc-18 homologue, not only causes the Sec-1 protein to sequester syntaxin from other proteins, but also To facilitate the interaction of syntaxin with other proteins by assisting them in adopting a functional conformation or by chaperone-like actions. The need for Sec1-related proteins is due, in part, to the direct and high-affinity interactions with members of the t-SNARE family of syntaxin proteins, as well as the v- and t-required by this complex. -It is considered to be controlled by SNARE protein interaction. The SNARE exocytosis machinery is evolutionarily conserved (most elements have homologues in species from yeast to mammals) and functional (all key elements are members of the multigene family). It is thought that The latter has been confirmed in studies showing the presence of elements of this pathway in several different cell types (neurons, neutrophils and β cells of the pancreas) (brumell J. et al.,-J immunol. -1995; 15
5: p5750-5759; Zhang W et al.,-J Biol Chem.-2000 Oct 6, electronic publi.
cation). Since the protein of SEQ ID NO: 392 belongs to the Unc-18 / Sec-1 family, it is thought to play an important role in controlling various processes such as vesicle targeting, docking and fusion.

One embodiment of the present invention is SEQ ID NO: 39 to identify fetal renal tissue and cells derived from this tissue because the protein of the invention is strongly expressed in this tissue.
2 or using the cDNA of SEQ ID NO: 151 or a part thereof. further,
The proteins of the invention can be used to specifically label components of the secretory pathway within cells. Techniques known to those of skill in the art can be used to develop assays to detect cells expressing the proteins of the invention or portions thereof. For example, the proteins of the invention or portions thereof can be used to raise antibodies or antisera using techniques known to those of skill in the art. The antibodies or antisera described above can be used for the quantitative analysis or detection of the proteins of the invention by methods such as enzyme-linked immunosorbent assay (ELISA), or any other technique known to those of skill in the art. As Sec1-related proteins are known to bind syntaxin, it is possible to use labeled syntaxin as an alternative. In other embodiments of the invention, the polynucleotides and polypeptides are allergic such as hay fever, asthma and urticaria; neurological disorders, some of which are associated with abnormal neurotransmitter secretion (eg, depression. The disease is associated with decreased serotonin secretion); autoimmune hemolytic anemia; cancers such as hormone-dependent cancers that specifically stimulate androgens (eg, prostate cancer) or estrogens (eg, breast cancer); leukemias or lymphomas; Ulcerative colitis; type 2 diabetes, sometimes associated with reduced insulin secretion; proliferative granulonephritis; inflammatory bowel disease; growth failure due to reduced growth hormone secretion; multiple sclerosis; myasthenia gravis; rheumatoid and bone Arthritis; scleroderma; Chediak-Higashi and Sjogren syndrome; systemic lupus erythematosus; thyroiditis;
Toxic shock syndrome; tissue injury due to trauma; viral, bacterial, fungal and protozoal infections; and other induced or otherwise abnormal vesicle transport and associated physiological or pathological disorders Can be used for diagnosing, treating, and / or preventing a number of diseases and disorders characterized by abnormal exocytosis, but not limited thereto. The relationship between the expression level and / or activity of the protein and the presence or absence of symptoms associated with abnormal vesicle transport such as the above disorders can be determined by, for example, Northern blot, Western blot, ELISA, or general in vitro or in vivo assay. Assays for protein activity can be easily assessed by assaying expression levels or activities and correlating the measured expression levels or activities with the presence or absence of disorders.
Disorders in which the protein of the invention is known to be associated as a positive factor include:
Assays for diagnosis or screening can be readily developed where elevated levels or activity of the protein can be indicative of the presence of the disease or the likelihood of the disease occurring. Also, such diseases or conditions are affected by disorders such as, but not limited to, antibodies, antisense oligonucleotides, dominant negative ones of proteins, and small molecule inhibitors that suppress protein expression or activity. By administering to a patient, the expression or activity of the protein can be suppressed and treated or prevented. Alternatively, for disorders in which the protein of the invention is associated as a negative factor, if the reduced level or activity of the protein indicates the presence of the disease or the likelihood of the disease occurring, diagnosis or screening of the disorder. Can be done easily. The disorders associated with the protein of the present invention as a negative factor from this fact include, for example, but not limited to, the protein itself, a polynucleotide encoding the protein, or a heterologous compound that increases the expression or activity of the protein. When administered to a patient, the level or activity of the protein can be increased to treat or prevent it.

<Protein of SEQ ID NO: 419 (Internal Designation Number 188-9-1-0-C10-CS)> The protein of SEQ ID NO: 419 highly expressed in the brain and placenta is the protein of SEQ ID NO: 178.
It is encoded by a cDNA, preferentially localized in the endoplasmic reticulum, and is homologous to the yeast integral membrane protein SFT2p (Genbank accession number X79489) belonging to the SNARE-related family. SFT2p was identified in C. elegans and mouse (accession numbers CAA93859 and AA79042, respectively).
It is well conserved in 5) and plays an important role in the protein transport and fusion mechanism of eukaryotic cells. The 159 amino acid long protein of the present invention is similar in size and membrane morphology to the SFT2p protein, with a conserved hydrophobic position at four positions 36-56, 66-86, 98-118 and 122-142. It shows a stretch and forms a tetraspan membrane protein.
Sch2p, as described by Conchon et al. In EMBO J., 18 (14): 3934-3946 (1999).
Another well-conserved SNARE-related protein, Got1p (P. falciparum, accession number AL010285; for C. elegans, accession number U23521), which has a function similar to that of the protein, also has this form. Eukaryotic proteins are synthesized in the endoplasmic reticulum (ER) and delivered to the Golgi complex for post-translational processing and sorting, from the Golgi complex to specific intracellular and extracellular destinations. Be transported. This movement of protein molecules inside and outside the cell is called vesicle transport. Transport is accomplished by budding from the membrane of the donor organelle and packaging the protein molecule in specialized vesicles that fuse with the target membrane (Palade, Science 189: 347-358 (1975)). Large numbers of proteins are required for the formation, targeting and fusion of transport vesicles and for the accurate sorting of proteins into these vesicles. The vesicle transport mechanism is a coat protein that promotes vesicle germination from the donor membrane, a vesicle- and target-specific identifier that binds to each other and docks the vesicles to the target membrane.
(v-SNARE and t-SNARE) (Nichols et al., Nature 387: 199-202, 1997), and SNAR
It contains a protein (SNAP) that binds to the E complex and initiates fusion of vesicles to the target membrane. SFT2p is a well-conserved yeast protein with four transmembrane domains that resides in a punctate structure corresponding to the late Golgi compartment and enters the putative retrograde Golgi vesicle, The vesicle fusion depends on two t-SNARE proteins, Sed5p and Sft1p (Wooding and Pelham, Mol. Biol. Cell 9: 2667-2680 (1998)). That Se
Genetic interaction with d5p suggests that SFT2p is an additional membrane element involved in the docking or fusion process. In vivo experiments are based on GOT1p or SFT2
Deletion alone does not affect cell growth, but when these proteins are suppressed together, the endoplasmic reticulum membrane accumulates significantly, so SFT2p or GOT1p can be maintained to maintain efficient endoplasmic reticulum-Golgi transport. Suggests the presence of either (Conchon
Et al., Supra). Got1p normally promotes Sed5p-dependent fusion events, while Sft
2p has been shown to perform related functions in late Golgi (Conchon et al., Supra).

The cause of many human diseases and disorders is believed to be due to defects in the transport of proteins to organelles or surfaces of cells. For example, defects in membrane-bound receptor and ion channel transport are associated with cystic fibrosis (cystic fibrosis transmembrane conductance regulator; CFTR), glucose-galactose malabsorption syndrome (Na + / glucose cotransporter), Hypercholesterolemia (low density lipoprotein (LDL) receptor
), And a type of diabetes mellitus (insulin receptor).
Abnormal hormone secretion is associated with disorders such as diabetes insipidus (vasopressin), hyper and hypoglycemia (insulin, glucagon), Graves' disease and goiter (thyroid hormone), Cushing and Addison's disease (adrenocorticotropic hormone; ACTH). Has been. Cancer cells also secrete hormones or other bioactive peptides in excess. Disorders associated with excessive secretion of bioactive peptides in tumor cells include islet cell adenoma-
Fasting hypoglycemia due to increased insulin secretion from islet cell tumor, adrenal medulla and)
Hypertension due to increased secretion of epinephrine and norepinephrine from pheochromocytomas in the parasympathetic ganglia; and excess vasoactive substances secreted from intestinal tumors
Includes abdominal cramps, diarrhea and heart valve disease caused by (serotonin, bradykinin, histamine, prostaglandins and polypeptide hormones).
Ectopic synthesis and secretion of bioactive peptides (unexpected peptides in tumors) is associated with ACTH and vasopressin in lung and pancreatic cancer; parathyroid hormone in lung and bladder cancer; calcitonin in lung and breast cancer; and medullary thyroid cancer. Including thyroid stimulating hormone. The protein of SEQ ID NO: 419 or a part thereof is considered to be an integral membrane protein of the SNARE-related family, and is presumed to be a human homologue of the yeast SFT2p protein, in particular. Thus, the proteins of the invention contribute to the secretion and endocytosis of eukaryotic cells in the transport and fusion of vesicles from the endoplasmic reticulum to late Gorgi systemna. Preferred polypeptides of the present invention are 36-56, 66-86, 98-118.
And a polypeptide comprising the amino acid sequence of SEQ ID NO: 419 with four transmembrane domains from positions 122-142. Other preferred polypeptides of the present invention are fragments of SEQ ID NO: 419 that include any of the biological activities described herein.

In one embodiment, the invention relates to methods and compositions using the proteins of the invention or parts thereof as novel marker proteins for selectively identifying secretion and endocytic transport, preferably It is applied to the identification of the transport in the endoplasmic reticulum, more preferably in the late Gorgi systemna. For example, using any technique known to those skilled in the art, the protein of the present invention or a part thereof can be detected using a specific antibody raised against the protein. Such organelle-specific antibodies can then be used to identify cells with unregulated transport systems, such as differentiated tumor cells, or to use immunochemistry to distinguish specific organelle types in tissue sections. Can be used. Furthermore, the proteins of the invention can be used to specifically identify brain and / or placenta cells, which are tissues in which the proteins are overexpressed. Another embodiment of the invention is to recombinantly or chemically fuse a fragment of the protein of the invention to a heterologous polypeptide or polynucleotide to bind a heterologous compound such as a polypeptide or polynucleotide to an endoplasmic reticulum, preferably an endoplasmic reticulum. It relates to a method of targeting late Golgi vesicles. Such a fusion protein has a cleavage site located between the sequence encoding the protein of the invention and the heterologous protein sequence in order to cleave the protein of the invention from the heterologous portion so that it can be purified. Can be designed as. Preferred fragments of proteins that can be used for such applications include the four transmembrane domains of the proteins of the invention or the endoplasmic reticulum or Golgi minor domains, as demonstrated by Conchon et al. And Wooding and Pelham, supra. Other fragments or parts thereof that carry organ targeting signals. Such heterologous compounds can be targeted to the secretory pathway to modulate endoplasmic reticulum-Golgi endocytosis and secretory activity. In one embodiment, the proteins of the invention screen peptide libraries for inhibitors of transport activity, which are detected by endoplasmic reticulum membrane or Golgi vesicle accumulation, as described by Conchon et al., Supra. Can be used for In yet another embodiment, the proteins of the invention are used to diagnose, prevent and / or treat a number of disorders that affect transport and / or fusion machinery, which disorders include cancer, cystic Fibrosis (membrane conductance regulator (CFTR) and CFTR-related membrane-bound receptors and ion channels in cystic fibrosis), glucose-galactose malabsorption syndrome (Na + / glucose cotransporter), hypercholesterolemia (low Density lipoprotein (LDL) receptor), and some types of diabetes mellitus
Endocrine, secretory, inflammatory and gastrointestinal disorders such as (insulin receptors); diabetes insipidus (vasopressin), hyperglycemia (insulin, glucagon), Graves' disease and goiter (thyroid hormone), Cushing and Addison Diseases associated with abnormal hormone secretion such as corticotropin (ACTH); pancreatic islet cell adenoma-fasting hypoglycemia due to increased insulin secretion from islet cell tumor, adrenal medulla, and chromaffin in the sympathetic paraganglia Excess vasoactive substances (serotonin, bradykinin, histamine, prostaglandins and polypeptides) secreted by hypertension and intestinal tumors due to increased secretion of epinephrine and norepinephrine from cell tumors.
Hormone-induced abdominal cramps, diarrhea and carcinoid syndrome including heart valve disease, but also disorders associated with the secretion of bioactive peptides from tumor cells in excess. Ectopic synthesis and secretion of bioactive peptides (unexpected peptides in tumors) is associated with ACTH and vasopressin in lung and pancreatic cancer; parathyroid hormone in lung and bladder cancer; calcitonin in lung and breast cancer; and medullary thyroid cancer. Including thyroid stimulating hormone.

The relationship between the expression level and / or activity of the protein and the presence or absence of a disorder associated with abnormal vesicle transport and / or secretion, such as those mentioned above, can be determined by, for example, Northern blot, Western blot, ELISA or Standard in vitro or in vivo methods for assaying protein activity can be used to readily assess expression levels or activities and correlate the detected expression levels or activities with the presence or absence of the disorder. For disorders in which the protein of the present invention is involved as a positive factor, a diagnostic or screening method can be easily developed when the elevated level or activity of the protein indicates the presence of the disease or the possibility of developing the disease. Also, such diseases or conditions include, but are not limited to, by administering antibodies, antisense oligonucleotides, dominant negative proteins and inhibitors of protein expression or activity, etc. to patients suffering from the disorder. , The protein expression or activity can be suppressed to treat or prevent. Alternatively, a disorder associated with a protein of the invention as a negative factor facilitates diagnostic or screening methods if the reduced level or activity of the protein indicates the presence of the disease or the likelihood of the disease occurring. Can be developed. Such disorders in which the protein of the present invention is associated as a negative factor include, but are not limited to, the protein itself, a polynucleotide encoding the protein, or a heterologous compound that increases the expression or activity of the protein. When administered to a patient, the level or activity of the protein can be increased and treated or prevented. Cancer cells also secrete hormones or other bioactive peptides in excess. Thus, in other embodiments, antagonists or inhibitors of the proteins of the invention may be administered to a patient to treat and / or prevent cancer by suppressing transport activity in transformed cells. Any cancer including, but not limited to, adenocarcinoma, sarcoma, melanoma, lymphoma and leukemia can be treated or prevented by this method. In a preferred embodiment, said cancer is of the digestive tract including prostate, pancreas, lung, tongue, brain, breast, bladder, adrenal gland, thyroid, liver, uterus, ovary, kidney, testis and small intestine, large intestine, rectum and stomach. Includes cancers such as glands, tissues and organs involved in the secretion or absorption of organs and the like. In a particular embodiment, antibodies specific for the proteins of the invention are used directly as antagonists or indirectly as a targeting or delivery mechanism to deliver drugs to cells or tissues expressing the proteins of the invention. it can. In addition, large amounts of the protein of the invention present in tumor cells can be used to control intracellular protein levels using, for example, specific antibodies detected by FACS, or other methods known to those of skill in the art to control. By comparison with cells, it can be easily used to diagnose or screen for cancer.

<Protein of SEQ ID NO: 297 (181-3-3-0-C9-CS)> The protein of SEQ ID NO: 297 encoded by the cDNA of SEQ ID NO: 56 is synaptogyrin 1 (Trembl). It is homologous to ID: Q9UGZ4). The proteins of the invention are strongly expressed in brain and fetal brain, fetal liver and testis. The protein of SEQ ID NO: 297 is a splice variant of synaptogyrin 1. The splicing of the cDNA of SEQ ID NO: 56 is different for exon 3; exon 3 of synaptogyrin 1 is 238 base pairs long, whereas SEQ ID NO: 56 is
Exon 3 has a length of 345 base pairs. This means frame shifting and
Induce a stop codon. Therefore, the protein of SEQ ID NO: 297 has amino acid 122
It is the same as synaptogyrin 1 and remains up to amino acid 122 including itself
The 22 amino acids are completely different. Compared to synaptogyrin 1, the proteins of the invention show the same N-terminal domain (highly conserved in all synaptogylins) and two of the four transmembrane helices. Preferred polypeptides of the present invention constitute the N-terminal cytoplasmic domain of the protein and are highly conserved among all members of the synaptogyrin family, amino acids 1-1.
6 (Kedra D et al., -Hum Genet.- 1998, 103 (2): 131-141).
Other preferred polypeptides of the invention are those containing amino acids 25-45 and / or 68-88, which constitute two transmembrane alpha helices. Therefore, the proteins of the present invention appear to be members of the synaptogyrin family. Synaptogyrins are closely related to proteins of the synaptophysin family, both involved in neurotransmission and, more generally, exocytosis and vesicle trafficking. Members of the synaptogylin family include synaptogylin 1 (including splicing variants 1a, 1b and 1c), cell gylin (ce
llugyrin, or synaptogyrin 2) and synaptogyrin 3 are included. Moreover, this protein family is evolutionarily conserved, as homologues of human synaptogyrin 1 have been detected in rat, mouse, and nematode. Synaptogyrin and synaptophysin are the most abundant vesicle elements, and together they account for over 10% of all vesicle membrane proteins. Despite seeming that synaptogyrin is dispensable for exocytosis itself (synaptogyrin and synaptophysin have apparently overlapping functions), they do not normally regulate exocytosis. Is essential to.

The normal function and organization of eukaryotic cells depends on the transport of diverse vesicles that selectively shuttle membranes and transporters between different compartments of the secretory and endocytic pathways. Several key proteins involved in membrane targeting and exocytosis have been identified and a key set of interactions has been defined, termed the SNARE (Soluble N-Ethylmaleimide Sensitivity Factor Attachment Protein Receptor) hypothesis Incorporated into the model (Rotheman J-Nature-1994, 372: p55-63). According to the SNARE hypothesis,
Vesicles are vesicles (v-SNAREs) and targets (t-SNAREs) that are a set of complementary membrane proteins.
) Binding to the target membrane through interaction with membrane proteins. Understanding vesicle transport was greatly facilitated by the characterization of synaptic vesicles in neurons. In exocytosis of synaptic vesicles, the vesicle proteins synaptobrevin and synaptogyrin (vesicle-associated membrane protein; also called VAMP) are v-SNARE
And the plasma membrane associated protein SNAP-25 (25 kDa synaptosomal associated protein) and syntaxin 1 function as t-SNAREs. Following formation of the SNARE complex (or core complex), the cytosolic proteins alpha, beta and gamma SNAP (soluble N-ethylmaleimide susceptibility factor attachment protein) and NSF (N-ethylmaleimide sensitive) are required for membrane fusion. Factor) gradually increases. In transgenic PC12 cells, synaptogyrin 1 and synaptophysin 1 were as effective as tetanus toxin light chain in suppressing exocytosis (Sugi
ta S. et al.,-J Biol Chem.-1999, 274 (27): 18893-901), suggesting that these proteins are strong regulators of exocytosis. More recently
It has been discovered that synaptogyrin has an important function in synaptic plasticity (Janz R. et al.- Neuron.-1999, 24 (3): 687-700). The cause of many human diseases and disorders is due to defects in the transport of proteins to organelles or cell surfaces. For example, defective transport of membrane-bound receptors and ion channels results in cystic fibrosis (cystic fibrosis transmembrane conductance regulator, CFTR).
Implicated in glucose-galactose malabsorption syndrome (Na + / glucose cotransporter), hypercholesterolemia (low density lipoprotein (LDL) receptor), and type of diabetes (insulin receptor) . Abnormal hormone secretion
Diabetes insipidus (vasopressin), hyperglycemia and hypoglycemia (insulin, glucagon)
, Graves' disease and goiter (thyroid hormone), Cushing's disease and Addison's disease (adrenocorticotropic hormone; ACTH) have been implicated in disorders.

In addition, cancer cells secrete excess amounts of hormones or other bioactive peptides.
Impairment associated with hypersecretion of bioactive peptides by tumor cells was increased by fasting hypoglycemia due to increased insulin secretion by islet cell tumors of islet cell adenomas; increased by adrenal medulla and sympathetic paraganglion pheochromocytoma Hypertension due to epinephrine and norepinephrine secretions; and abdominal cramps, diarrhea, caused by secretion of excess vasoactive substances (serotonin, bradykinin, histamine, prostaglandins, and polypeptide hormones) by intestinal tumors
And carcinoid syndrome including valvular heart disease. Bioactive peptides (unexpected in tumors) are the synthesis of ACTH and vasopressin in lung and pancreatic cancer, parathyroid hormone in lung and bladder cancer, calcitonin in lung and breast cancer, and thyroid-stimulating hormone in medullary thyroid cancer. And secretion. In one embodiment, the invention relates to methods and compositions using the proteins of the invention or parts thereof as novel marker proteins to selectively identify secretion and endocytic transport, preferably endoplasmic reticulum. , More preferably for identification in the late Golgi cistern. For example, a protein of the invention, or a portion thereof, can be detected using specific antibodies raised against the protein using any technique known to those of skill in the art. Such organelle-specific antibodies can then be used to identify cells with a disrupted transport system, such as differentiated tumor cells, or to identify specific organelles in cell cross sections using immunochemistry. . In addition, the proteins of the invention can be used to specifically identify cells of the brain, fetal brain, fetal liver and testis, which are tissues in which the proteins are overexpressed. In another embodiment of the invention, a heterologous compound such as a polypeptide or polynucleotide is constructed into a secretory machinery by recombinantly or chemically fusing a fragment of the protein of the invention to the heterologous polypeptide or polynucleotide. Compositions and methods for targeting agents. Such a fusion protein is engineered to contain a sequence encoding the protein of the invention and a cleavage site located between the heterologous protein sequences, allowing the protein of the invention to be cleaved from the heterologous moiety and purified. Good. Such heterologous compounds may target the secretory pathway to modulate ER-Golgi endocytosis and secretory activity. In one embodiment, the proteins of the invention are used to screen peptide libraries for inhibitors of transport activity detected by ER membrane or Golgi vesicle accumulation, as described in Conchon et al., Supra. Can be used.

In yet another embodiment, the proteins of the invention are cancer, cystic fibrosis (cystic fibrosis transmembrane conductance regulator; CFTR, and membrane-bound receptors and ion channels associated with CFTR), glucose-. Galactose malabsorption syndrome (Na + / glucose cotransporter), hypercholesterolemia (low density lipoprotein (LDL) receptor), and type of diabetes (insulin receptor), diabetes insipidus (vasopressin), hyperglycemia and low Increased due to disorders associated with abnormal hormone secretion including blood glucose (insulin, glucagon), Graves' disease and goiter (thyroid hormone), Cushing's disease and Addison's disease (adrenocorticotropic hormone; ACTH), islet cell tumor of islet cell adenoma Fasting hypoglycemia, adrenal medulla and sympathetic paragans due to insulin secretion Hypertension due to increased epinephrine and norepinephrine secretion due to lion pheochromocytoma, abdominal cavity caused by intestinal tumor secretion of excess vasoactive substances (serotonin, bradykinin, histamine, prostaglandins, and polypeptide hormones) Including endocrine, secretory, inflammatory, and gastrointestinal disorders such as disorders associated with hypersecretion of bioactive peptides by tumor cells, including carcinoid syndromes, including convulsions, diarrhea, and valvular heart disease, It is used to diagnose, prevent and / or treat any of a number of disorders in which transport and / or fusion mechanisms are affected.
Ectopic synthesis and secretion of bioactive peptides, peptides that are not expected to be produced in tumors, are associated with ACTH and vasopressin in lung and pancreatic cancers, parathyroid hormone in lung and bladder cancers, calcitonin in lung and breast cancers, and thyroid. Includes thyroid stimulating hormone synthesis and secretion in medullary cancer. Linking the level of expression and / or activity of the protein with any pathological condition associated with the presence or absence of any abnormal vesicle trafficking and / or secretion such as the disorders described above may be performed, for example, on Northern blots, Western blot, ELISA
, Or by detecting the level of protein expression and / or activity, such as by standard in vitro or in vivo assays that measure protein activity, and correlating the measured level of expression and / or activity with the presence or absence of a disorder. ,
It can be easily determined. For disorders found to be positively associated with a protein of the invention, detection of elevated levels of the protein or protein activity is indicative of the presence or propensity for the disease and is therefore useful for diagnosis or screening. The assay can be easily developed. In addition, any such disease or condition may be impaired by inhibitors, including, but not limited to, antibodies, antisense oligonucleotides, dominant negatives of proteins, and small molecule inhibitors of protein expression or activity. It can be treated or prevented by administering to a patient suffering from the disease and suppressing the expression or activity of the protein. Alternatively, in a disorder negatively associated with a protein of the invention, a decrease in the level of protein or protein activity indicates the presence of disease or a tendency to develop disease, so detection of the level of protein or protein activity is Can be diagnosed or screened. Such disorders negatively associated with the protein of the invention include, but are not limited to, a plurality of, including, but not limited to, the protein, a polynucleotide encoding the protein, or a heterologous compound that enhances expression or activity of the protein. Administer any of the formulations of
It can be treated or prevented by increasing the level of protein or protein activity.

Cancer cells secrete excess amounts of hormones or other bioactive peptides. Therefore, in other embodiments, antagonists, inhibitors or other modulators of the proteins of the invention can be administered to a subject to treat or prevent cancer by suppressing transport activity in transformed cells. in this way,
Any type of cancer can be treated or prevented, including but not limited to adenocarcinoma, sarcoma, melanoma, lymphoma, and leukemia. In a preferred embodiment, the cancer comprises glands, tissues, and organs involved in secretion and absorption, including prostate, pancreas, lungs, tongue,
Brain, breast, bladder, adrenal gland, thyroid, liver, uterus, ovary, kidneys, testicles, and small intestine,
Includes organs belonging to the gastrointestinal tract, such as the colon, rectum, and stomach. In one particular embodiment, an antibody specific for a protein of the invention directly binds a substance that has been dispensed to a cell or tissue that expresses the protein of the invention, directly as an antagonist. It can be used as a targeting or delivery mechanism for locomotion. In addition, the protein is associated with depression, such as depression associated with decreased serotonin secretion,
The content of neurotransmitter release from one or more neural cell types, to diagnose, treat or prevent any neurological or psychological disorder or disease associated with abnormal neurotransmitter release, Neurological functions, such as memory, that can be enhanced or modulated by changing either frequency or other characteristics,
It can be used for diagnosis, treatment or prevention. <Proteins of SEQ ID NOs: 247 and 246 (internal designation numbers 105-031-2-0-D3-CS and 105-031-1-0-A2-CS)> Encoded by the cDNAs of SEQ ID NOs: 6 and 5, respectively. SEQ ID NOs: 247 and 2
46 proteins are overexpressed in liver, pancreas and prostate. The protein of the present invention shows strong homology to human membrane-bound protein PRO836 (GENSEQP accession number: W63687) and human secretory protein 7 (GENSEQP accession number: Y57941). Furthermore, the protein of the present invention has 27% identity with amino acids 68 to 340 of the protein of SEQ ID NO: 247, and has homology with SLS1p (GENPEPT accession number Z50154), which is a chaperone-associated protein detected in yeast Yarrowia lipolytica. To share. Furthermore, the proteins of SEQ ID NOs: 247 and 246 are mouse (GENSEQP accession number: Z50154).
And Hsp binding protein 1 detected in human (GENPEPT accession number: AF093420),
And Hsp binding protein 2 detected in human (GENPEPT accession number: AF187859),
Shares homology with two proteins of the Hsp70 family of.

The proteins of the present invention relate to the endoplasmic reticulum yeast luminal protein, SLS1p. This protein acts in a pre-protein translocation process to interact directly with translocation polypeptides, facilitate their migration and / or aid their folding in the endoplasmic reticulum (Boisrame et al., J Biol Ch
em 1996; 271: 11668-75). Furthermore, Sls1p appears to act as a cofactor for the chaperone protein Kar2 (Boisrame et al., J Biol Chem 1998; 273: 30903-8).
Kabani et al., Gene 2000; 241: 309-15). Therefore, the protein of the present invention is presumed to have a cell function similar to that of chaperone. Such functions include several cellular processes such as protein folding, degradation of oligomeric protein structure, regulation of apoptosis, protein degradation, protein translocation to the endoplasmic reticulum, and antigen presentation (Bukau et al. Cell 1998; 92: 351-66). In addition, chaperones have been implicated in multiple disorders, particularly autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, and mixed connective tissue disease (Feige et al., J Autoimmun 2000; 14: 133-42). Furthermore, the chaperone
Neurogenic disorders such as tuberculosis and leprosy (Zugel et al., Clin Microbiol Rev 1999; 12: 19-39), Alzheimer's disease and Parkinson's disease (Yoo et al., J Neural T
ransm Suppl 1999; 57: 315-22), and malignant disorders (Csermely et al., Pharmacol Th.
er 1998; 79: 129-68). Moreover, increasing evidence suggests that the Hsp60 chaperone is involved in the development of atherosclerosis (Xu
Circulation 2000; 102: 14-20). Therefore, as summarized above, the present protein, which is presumed to be a co-factor of chaperone, has a cellular function similar to that of chaperone and seems to be involved in similar pathological processes. In one embodiment, the invention provides a method of using the protein to identify specific cell types in vivo and in vitro. For example, chaperone proteins are often upregulated in response to cellular stress, so detection of cells expressing elevated levels of the protein would provide a means of detecting cells under stress. Since cellular stress has been implicated in several disorders such as cardiovascular disorders, neurodegenerative disorders, and cancer, the ability to detect such stress is therefore a diagnostic or screening tool for such disorders. I will provide a. In addition, the polypeptides and polynucleotides can be used to identify liver tissue, pancreatic tissue and prostate tissue, and cells derived from these tissues. The ability to specifically visualize tissues and cells facilitates determining the origin or identity of, for example, cancer cells, and identification of specific cells and tissues, eg, for histological slide evaluation. It is useful for many applications, including

In addition, the polypeptides and polynucleotides can be used to develop diagnostic and screening assays for diseases that are characterized by abnormal levels or activity of the proteins of SEQ ID NOs: 247 and 246. Such disorders include infectious diseases, neurogenesis disorders such as Alzheimer's disease and Parkinson's disease, schizophrenia, alopecia,
It includes, but is not limited to, aging, atherosclerosis, various types of malignant disorders, and autoimmune diseases including type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, and mixed connective tissue disease. Such an assay can be performed by using any biological sample such as serum or plasma. In yet another embodiment, the proteins of the invention, or portions thereof, can be used to prevent cells from undergoing apoptosis. Specifically, since it is clear that chaperone proteins protect cells from apoptosis, any method of increasing the level or activity of the protein can be used in vitro or in vivo to prevent cells from undergoing apoptosis. . For example, SEQ ID
Either the polynucleotide encoding the 247 or 246 protein, or a fragment or derivative thereof, can be introduced into a cell, eg, a vector in which the protein is expressed in the cell. Alternatively, SEQ ID NO: 247 or 246
The protein itself can be administered to the cell, preferably in a dosage form that reaches the internalization of the protein by the cell. In addition, any compound that increases protein expression or activity in the cell can be administered. Preventing cells from undergoing apoptosis is the prevention of growing cell death in culture samples, the prevention of apoptosis associated with multiple disorders in patients, or a treatment that increases the level of cellular stress such as chemotherapy. It may be utilized to serve any of a number of purposes including, but not limited to, preventing apoptosis in patients undergoing treatment. In other embodiments, inhibiting the proteins of the invention can be used to induce apoptosis in unwanted cells. Such suppression can be performed by any of a number of methods including, but not limited to, using antibodies, antisense sequences, dominant negatives of proteins, or small molecule inhibitors of protein expression or activity. Induction of such apoptosis can be utilized to eliminate any unwanted cells in the patient, such as cancer cells. Preferably, such inhibitors are those that specifically target unwanted cells in the patient.

In another embodiment, either by the protein of SEQ ID NO: 247 or 246, or a portion thereof, or by other compounds capable of affecting the level or activity of these proteins, such as nucleic acids, antibodies, chemicals, and the like. , Can treat, alleviate and / or prevent a variety of disorders. In a preferred embodiment, proteins or other compounds directed against the proteins of the invention can be used to treat or prevent disorders in which the activity or level of the protein of SEQ ID NO: 247 or 246 is disproportionate. Such diseases include infectious diseases, neurogenesis disorders such as Alzheimer's and Parkinson's disease, schizophrenia, alopecia, aging, atherosclerosis, various types of malignant disorders, and type 1 diabetes, rheumatoid arthritis, systemic disease. Includes, but is not limited to, autoimmune diseases including lupus erythematosus, Sjogren's syndrome, and mixed connective tissue disease, malignant disorders, autoimmune and other neurodegenerative disorders. In another embodiment, the protein of SEQ ID NO: 247 or 246 or a portion thereof is cancer (Wang et al. Immunol Invest).
2000; 29: 131-7), tuberculosis (Silva et al., Microbes Infect 1999; 1: 429-35), diabetes (Int Immunol 1999; 11: 957-66), and atherosclerosis (Xu et al., Arterioscle).
r Thromb 1992; 12: 789-99) and can be used as a vaccine for a variety of disorders, including but not limited to. <Protein of SEQ ID NO: 389 (Internal Designation Number 109-003-1-0-G4-CS)> The protein of SEQ ID NO: 389 is encoded by the cDNA of SEQ ID NO: 148. Accordingly, all features and uses of the polypeptide of SEQ ID NO: 389 described elsewhere in this application also relate to the polypeptide encoded by the human cDNA which is clone 109-003-1-0-G4-CS. It will be understood. Furthermore, all the features and uses of the nucleic acid of SEQ ID NO: 148, described elsewhere in this application, also apply to the nucleic acid encoded by the human cDNA, clone 109-003-1-0-G4-CS. It will be understood that this is the case. The protein of SEQ ID NO: 389 exhibits a high degree of homology to two human proteins encoded by the genes listed under accession numbers AF143723 and AF112210 in Genbank, the disclosures of which are fully incorporated herein by reference. Transferred. The polypeptides encoded by Genbank accession numbers AF143723 and AF112210 belong to the Hsp70 protein family (although one of them was mistakenly said to belong to the related Hsp60 family). For example, human hsp70 (GenBan
Accession numbers M11717 and M15432; also Hunt and Morimoto, 1985, Proc. Natl.
Acad. Sci. USA 82: 6455-6459, the disclosures of which are incorporated herein by reference in their entirety, human hsp90 (GenBank Accession No. X15183;
See Yamazaki et al., 1989, Nucleic Acids Res. 17: 7108; their disclosures are incorporated herein by reference in their entirety), and human gp96 (Ge.
nBank Accession No. M33716; also Maki et al., 1990, Proc. Natl. Acad. Sci. USA 87:
5658-5662, the disclosures of which are incorporated herein by reference in their entirety), many genes encoding "Hsp" (heat shock protein) have been cloned and sequenced. Was done.

The protein of SEQ ID NO: 389 and the two homologues of the above are indeed more than the human Hsp70, the corresponding gene for a previously unidentified human member of the family,
Close to the yeast members of the family. Both the Hsp70 signatures of Pfam and Prosite ("HSP70" Pfam model at amino acids 3-509 and PS01036 Prosite motif at positions 332-346, respectively) are recognized within the protein of SEQ ID NO: 389.
The protein of SEQ ID NO: 389 is different from the protein encoded by AF112210 in
It differs at amino acid positions 282, 312 and 326, and differs from the protein encoded by AF143723 at amino acid positions 15 and 326. Heat shock proteins are molecular chaperones previously known to play a number of important roles in intracellular and intercellular processes, including protein synthesis and folding, vesicle trafficking, and antigen processing and presentation. It is a family of proteins. Hsp is one of the most highly conserved proteins and carries out many regulatory activities via protein-protein interactions. Historically,
These have been identified by induction under stress conditions, and are now known to provide the essential action of preventing aggregation of misfolded proteins and supporting refolding. The major stress proteins accumulate to very high levels in stressed cells, but are present at low to moderate levels in unstressed cells. Hsp70 is a member of the heat shock protein family. (Milner, CM
and Campbell, RD Immunogenetics 32: 242-251 (1990); GenBank Accession No. M59828, the disclosures of which are incorporated herein by reference in their entirety). The 70 kD heat shock protein is a highly conserved, widely distributed protein involved in escorting proteins to diverse organelles. It is highly evoked in mammals, as opposed to other members of the Hsp family. Hsp70 is one of the most actively synthesized proteins in cells upon heat shock, although it is barely detectable at normal temperature (Welch et al., 1985, J. Cell. Biol. 101: 1198-. 1211, the disclosure of which is incorporated herein by reference in its entirety). In contrast, Hsp90 and Hsp
The 60 protein is abundant in most, if not all, mammalian cells at normal temperature and is heat-induced (Lai et al., 1984, Mol. Cell. Biol. 4: 2802-10.
Van Bergen en Henegouwen et al., 1987, Genes Dev., 1: 525-31, each publication incorporated by reference in its entirety). Furthermore, the Hsp70 protein acts as a monomer, but the functionally related Hsp60 proteins are associated in vivo in large macrocycles close to one million daltons. All the various actions of Hsp
Basically, ATP is complexed with a polypeptide segment, preferably one that is hydrophobic.
Dependently, it is based on its ability to stabilize in an extended conformation. Complexed polypeptides can be antigenic peptides (where Hsps help guide them to the major histocompatibility complex for presentation) or repeated free / refolding cycles following binding with Hsps. May be a misfolded protein that facilitates the proper conformation by (Bukau, B. and Horwich L., 1998, Cell 92.
: See 351-366. The publication is incorporated herein by reference in its entirety).

Based on the above information, the protein of SEQ ID NO: 389 appears to be a member of the human Hsp70 family. Thus, the protein of SEQ ID NO: 389 may play a role in protein synthesis / folding, cell trafficking, antigen processing, cellular stress response and immune response in immunocompetent cell types. Other information regarding the protein of SEQ ID NO: 389 is described in Rudiger et al., 1997, EMBO J. 16, 1501-1507, the disclosure of which is incorporated herein by reference in its entirety. Can be used to obtain binding assays using a common Hsp70 substrate. In one embodiment of the invention, the protein of SEQ ID NO: 389, or contiguous amino acids thereof, is at least 5, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150 or A fragment containing 20 or slowing the degradation of protein in an individual,
It relates to a method of using a fragment having a desired biological activity as an immobilized adjuvant for enhancing the yield of a recombinant protein or regenerating a denatured protein. In such an embodiment, the protein of SEQ ID NO: 389 of the fragment is a composition comprising a protein in which it is desired to slow degradation, enhance yield, or regenerate denatured protein under conditions that facilitate obtaining the desired results. Mix with things. For example, many commercially available assay kits commonly used by those skilled in the field of molecular biology and biochemistry rely on the biological properties of proteins (mainly enzymes), and due to the low stability of these proteins, It may be very short lived in vitro. In its publication DE 4124286, the low intrinsic stability of the test solutions used for optical tests is increased by the addition of chaperone proteins, the test of which is therefore incorporated in its entirety by reference. An example has been described in which is more sensitive. In addition, the protein of SEQ ID NO: 389 can be used to increase recombinant protein yield or activity. The main unsolved problems in recombinant DNA technology are:
Solubility and biological activity of recombinantly overexpressed proteins in a host, especially a bacterial or yeast host. Many eukaryotic proteins, especially those secreted, are deficient in bacterial hosts such as E. coli due to correct folding, or deficient in mammalian / yeast cells due to high expression of the protein,
Requires specific cell machinery. The ability of the protein of SEQ ID NO: 38, or fragments thereof, to ensure proper folding of recombinant proteins can be used as follows. The protein of SEQ ID NO: 389 may be co-expressed with a recombinant protein in a bacterial or eukaryotic host so that the host expresses the heterologous protein or polypeptide in a form with increased solubility and / or biological activity. . For example,
The protein of SEQ ID NO: 389 or a fragment thereof can be used in the method of International Patent Application WO 93/25681, the publication of which is incorporated herein by reference in its entirety. Alternatively, the protein of SEQ ID NO: 389 or a fragment thereof is incorporated by reference in its international patent application WO 00/0813, the disclosure of which is incorporated herein by reference in its entirety.
It may be added exogenously to the cell culture as described in 5. In fact, the international patent application WO 00/31113, the entire disclosure of which is incorporated herein by reference,
Show that when exogenously added to cells, Hsp70 is immediately imported into both the cytoplasmic and nuclear compartments. Preparation and purification of the protein of SEQ ID NO: 389 or fragments thereof may be carried out as described in patent US-6,007,821, the disclosure of which is incorporated herein by reference in its entirety.

Further, the protein of SEQ ID NO: 389 or fragments thereof can be used to regenerate denatured proteins. Recombinantly expressed, poorly bioactive proteins are usually denatured with strong denaturants such as guanidine hydrochloride, followed by refolding by dilution with large volumes of diluent to reduce the concentration of denaturants. However, although this method frequently reaches poor refolding rates, a cocktail of chaperone proteins is obtained in a manner similar to that described for Hsp60 in European Patent EP0650975, the disclosure of which is incorporated herein by reference in its entirety. Can be significantly increased by adding. The advantage of using a cocktail of chaperone proteins is to accommodate the different binding specificities of different families of Hsps and different members within each family. For example, vertebrate actin is efficiently folded by the eukaryotic cytosolic chaperonin (Gao et al., 1992, Cell 69: 1043-1050, the publication of which is incorporated herein by reference in its entirety). Does not fold in the case of Hsp60 (Tian et al., 1995, Nature
375: 250-253, the entire text of which is incorporated herein by reference). Other embodiments of the present invention include heterologous compounds (proteins, peptides, or DNA).
SEQ ID NO: 38 for the delivery of a) to specific cell compartments, preferably the cytoplasm and nucleus.
9 uses of the protein or fragments thereof. If desired, the protein of SEQ ID NO: 389 or a fragment thereof can be fused to a heterologous compound. For example, the protein of SEQ ID NO: 389 or a fragment thereof, the disclosure of which is incorporated herein by reference in its entirety in International Patent Application WO 00/31113.
It can be used to attend compounds into cells using the methods described in.
In the method described in WO 00/3113, Hsp70 was used to deliver NF-KB, a key transcriptional regulator of inflammatory responses, to the nuclear compartment. C-terminal Hsp70 peptide and N
It was demonstrated that a fusion protein consisting of amino acids 37-409 of the p50 subunit of F-KB was directed to the cell nucleus and specifically bound to DNA, activating κIg expression and TNFa production. In one embodiment of the invention, the protein of SEQ ID NO: 389 or a fragment thereof can be used in human therapy as a modulator of the immune response. Hsp70 of the present invention,
Pathological conditions that can be treated by the fragment, and / or the Hsp70 complex, are transplant rejection (see US Pat. No. 5,891,653; the publication of which is incorporated herein by reference in its entirety) and insulin dependent. Full text of autoimmune diseases such as diabetes mellitus, rheumatoid arthritis, multiple sclerosis, juvenile urinary disease, asthma, and inflammatory bowel disease, as well as inflammatory diseases, cancers, viral replication diseases, and their respective publications. Including the vascular diseases described in the following patents incorporated by reference:
US6,007,821; WO 00/31113; WO 99/18801 (Treatment of autoimmune diseases), US6,017,540
US6,017,544; AU3425899; WO 99/54464; US5,837,251; US5,830,464; WO 98/3
4642; WO 98/34641; US5,750,119; WO 97/10001; WO 96/10411 (treatment of cancer); DE1
9813760, DE19813759 (both autoimmune diseases and cancer).

The protein of SEQ ID NO: 389 or a fragment thereof may be used in the treatment of autoimmune diseases,
Or can be used for improvement. In this embodiment, the composition of the heat shock / stress protein complex, including but not limited to the protein of SEQ ID NO: 389, is administered to an individual suffering from an autoimmune disease. The complex may be composed of a single protein of SEQ ID NO: 389 or a fragment thereof, or may include other heat shock / stress proteins. In one embodiment, the protein of SEQ ID NO: 389 or a fragment thereof is non-covalently bound to the antigen molecule and is administered to an individual suffering from an autoimmune disease in order to suppress the autoimmune response. Alternatively,
The protein of SEQ ID NO: 389 or a fragment thereof is in uncomplexed form (ie,
A composition comprising (without an antigenic molecule) can also be administered to an individual suffering from an autoimmune disease in order to suppress the immune response (see US Pat. No. 6,007,821; the publication of which is hereby fully incorporated). Incorporated in the description as a reference). The ability of stress proteins to associate with the antigenic peptides of the cells from which they are derived can be used to isolate antigenic peptides expressed in tumors. In this embodiment of the invention, a complex consisting of the protein of SEQ ID NO: 389 or a fragment thereof, and an antigenic peptide expressed by the tumor is isolated. The isolated complex is re-administered to the individual who obtained the complex to elicit an immune response against the tumor. Thus, this method avoids the need to isolate and characterize specific tumor antigens and allows one of skill in the art to readily prepare immunogenic compositions effective against tumors in individuals. (See US Pat. No. 6,017,544; the disclosure of which is incorporated by reference in its entirety). In addition, the protein of SEQ ID NO: 389 can be used to diagnose bladder cancer. The segment extending from amino acid position 1 to 187 of the protein of SEQ ID NO: 389 is 99% or more identical to a polypeptide associated with bladder cancer. (See European Patent DE 19818620; the publication of which is incorporated herein by reference in its entirety). The 187 amino acid long polypeptide described in DE19818620 was identified as the only partial product of a gene whose expression in bladder tumors is significantly altered compared to healthy bladder. In another embodiment of the invention, the protein of SEQ ID NO: 389 or a fragment thereof can be used to diagnose disorders associated with aberrant intercellular communication or secretion. In such methods, the protein level of SEQ ID NO: 389 in diseased individuals is measured using methods such as those described herein. The protein level of SEQ ID NO: 389 in diseased individuals is compared to the level in healthy individuals. The altered level of SEQ ID NO: 389 compared to a healthy individual suggests that the individual has bladder cancer. The level of protein of SEQ ID NO: 389 present in an individual can be determined by contacting a sample from the individual with an antibody raised against the polypeptide of SEQ ID NO: 389. Alternatively, the protein level of SEQ ID NO: 389 in the individual may be measured by determining the RNA level encoding the protein of SEQ ID NO: 389 in the test sample. RNA levels can be measured by nucleic acid array or in situ hybridization, Northern blot,
It can be measured using a method such as dot blot or other methods known to those skilled in the art. If desired, the nucleic acid sample may be subjected to an amplification reaction, such as a PCR reaction, prior to analysis. The RNA level in the sample is compared with the RNA level in a healthy individual to determine whether the individual has bladder cancer.

In addition, the publication is described in US Pat. No. 5,188,964, which is incorporated herein by reference in its entirety, as an antibody against the protein of the protein of SEQ ID NO: 389, or SEQ ID NO: Nucleic acid probes complementary to the sequence encoding the 389 protein can also be used as a prognosis for tumor regression in the breast. Specific levels of stress response proteins (including Hsp70) have been identified, as described in US Pat. No. 5,188,964, above which the rate of tumor recurrence becomes quite significant. Therefore, in a sample of individuals who have previously experienced breast tumors, the protein of SEQ ID NO: 389 or the RN encoding the protein of SEQ ID NO: 389
Determine the level of A. Protein or RNA levels may be measured as described herein. If the protein or RNA level is above the level above which tumors are likely to develop, a course of appropriate treatment may be initiated. In another embodiment of the invention, the protein of SEQ ID NO: 389 is capable of tissue repair under stress conditions such as hypoxia, oxidative stress, genotoxic agents, and more generally, deleterious conditions that result in programmed cell death. And / or increase cell survival. The beneficial effects are exerted either by protecting cellular proteins from premature denaturation / degradation or by directly suppressing signaling pathways leading to programmed cell death (Gabai VL. Et al., 1998, FEBS Lett. 438). : 1-4, the entire publication is incorporated herein by reference). These conditions include, but are not limited to, infarction, heart surgery, stroke, neurodegenerative disease, epilepsy, trauma, atherosclerosis, post-restenosis angiogenesis, and nerve injury. For example, hypoxic stress is known to be a signal that increases the amount of Hsp70 in heart tissue, where Hsp70 binds to partially denatured proteins and leads to a more stable native structure of the protein. By assisting in the refolding of cells, it contributes to the survival of cells. Such aids provide very important cardioprotection during periods of hypoxia, such as during infarction or surgery where blood flow to the heart may be temporarily interrupted. In addition, numerous research groups have investigated Hsp70 in different models of nervous system injury.
Has been shown to be over-produced in protection (Midori AY et al., 1999, Mol.
Med.Today, 5: 525-31; its publication is incorporated herein by reference in its entirety). Therapeutic methods for administering the protein of SEQ ID NO: 389 or a fragment thereof include those disclosed in patent WO 00/23093, the disclosure of which is incorporated herein by reference, Not limited to.

Accordingly, it may be desirable to increase or decrease the level of the protein of SEQ ID NO: 389 in an individual having a disease that results from the increase or decrease of the level of the protein of SEQ ID NO: 389. In such an embodiment, SEQ ID NO: 38
The 9 proteins, or fragments thereof, are administered to an individual in whom it is desired to increase or decrease any of the above activities. The protein of SEQ ID NO: 389 or fragment thereof may be administered directly to the individual, or the nucleic acid encoding the protein of SEQ ID NO: 389 or fragment thereof may be administered to the individual. Alternatively, a formulation that increases the protein activity of SEQ ID NO: 389 may be administered to the individual. Such formulations can be identified by contacting the protein of SEQ ID NO: 389 or cells or preparations containing the protein of SEQ ID NO: 389 with a test agent and assaying whether the test agent increases the activity of the protein. For example, the test agent can be a chemical compound or polypeptide or peptide. Alternatively, the protein activity of SEQ ID NO: 389 can be reduced by administering to the individual a formulation that interferes with such activity. A formulation that interferes with the activity of the protein of SEQ ID NO: 389 is to test whether the test agent reduces protein activity by contacting the protein of SEQ ID NO: 389 or a cell or preparation containing the protein of SEQ ID NO: 389 with a test agent. Can be identified by For example, the formulation may be a chemical compound, polypeptide or peptide, antibody, or nucleic acid such as an antisense nucleic acid or a triple helix forming nucleic acid. <Protein of SEQ ID NO: 250 (Internal Designation No. 105-053-4-0-E8-CS)> The protein of SEQ ID NO: 250 is encoded by the cDNA of SEQ ID NO: 9. All features and uses of the polypeptide of SEQ ID NO: 250, described elsewhere in this application, also apply to the polypeptide encoded by the human cDNA which is clone 105-053-4-0-E8-CS. It will be understood that this is the case. Furthermore, all the features and uses of the nucleic acid of SEQ ID NO: 9 described elsewhere in this application also apply to the nucleic acid encoded by the human cDNA, clone 105-053-4-0-E8-CS. It will be understood that this is the case. The protein of SEQ ID NO: 250 was detected in the prostate and exhibits extensive homology with the range of pancreatic zymogen granulosa membrane protein GP2 (glycoprotein 2). In particular, the protein of SEQ ID NO: 250 is human (SWISS-PROT accession number P55259, the publication of which is incorporated by reference in its entirety), rat (SWISS-PROT accession number P19218, the publication of which is fully Are incorporated herein by reference) and dogs (SWISS-PROT accession number P25291, the disclosure of which is incorporated herein by reference in its entirety) and show homology with the above-mentioned GP2 proteins. . In fact, the protein of SEQ ID NO: 250
The amino acid sequence of SEQ ID NO: 250 is an exact match to the amino acid sequence of the human GP2 sequence, except that it lacks amino acids 62-484 as compared to the 2 sequence. The protein of SEQ ID NO: 250 contains two hydrophobic regions, an N-terminal signal peptide (amino acid residues 8-28) and a C-terminal transmembrane region (amino acid residues 91-111).

GP2 (glycoprotein 2) is the major membrane glycoprotein of secretory zymogen granule (ZG) membranes in pancreatic acinar cells (Fukuoka et al., 1990 Nuc. Acids Res., 18: 5900; Fu).
kuoka et al., 1991 Proc. Natl. Acad. Sci., USA, 88: 2898-2902; Fukuoka et al., 199.
2 Proc. Natl. Acad. Sci. USA, 89: 1189-1193; Freedman et al., 1993 Eur. J. Cel.
Biol. Scheele et al., 1993 Pancreas: 139-149; Freedman et al., 1994 Annals NY.
Acad. Sci. 713: 199-206, each publication incorporated by reference in its entirety). GP2 homologues are widely distributed among diverse epithelial tissues known to have controlled secretory processes, including parotid, submandibular gland, stomach, liver and lung (Fuku.
oka et al., 1992 Proc. Natl. Acad. Sci. USA, 89: 1189-1193) In addition to the ZG membrane, GP2 also contains rough endoplasmic reticulum, Golgi, trans-Golgi elements, concentrated vacuoles and apical apical cells of pancreatic acinar cells. Basolateral plasma membrane (APM), basolateral plasma membrane (BPM), and Z
It is located within the G and acinar lumen (Scheele et al., 1994 Pancreas 9: 139-149). GP2 is bound to the membrane of ZG via a glycosylphosphatidylinositol anchor (GPI anchor) (Fukuoka et al., 1991 Proc. Natl. Acad. Sci. USA, 88: 2898).
-2902; Lebel and Beattie 1988 Biochem. Biophys. Res. Comm. 254: 1189-93,
The disclosures of them are incorporated by reference herein in their entirety), pH about 6.5.
Below, a complex, usually a tetrameric complex, is formed. GP2 during secretory granule assembly during trans-Golgi network (TGN)
The complex is formed by the low pH of TGN. These complexes bind proteoglycans (PG) to form a fibrillar GP2 / PG network on the luminal surface of ZG. The GP2 / PG matrix is capable of functioning in membrane sorting within the TGN, assembly of ZG membranes, inactivation of ZG membranes during granule storage, and control of ZG membrane transport at the apical plasma membrane. is there. Furthermore, the GP2 / PG matrix protects the luminal surface of the granulosa from contact with secretory enzymes contained in the granules,
It may facilitate the specific release of secreted enzymes during exocytosis at the apical plasma membrane. The enzyme and acidic environment contained in the ZG are released by acinar cells via exocytosis into the lumen of the pancreas. The pH in acinar cells and the apical plasma membrane of the general pancreatic lumen is maintained at an essentially neutral or alkaline pH by fluids and bicarbonate secreted by pancreatic duct cells. The increase in pH at the apical plasma membrane (relative to the acidic pH in ZG) optimizes the enzymatic cleavage conditions for GP2 GPI anchors, which results in the removal of GP and GP2 / PG complexes from the apical membrane. discharge. (Scheele et al., (1994) Pan
creas 9: 139-149, the entire disclosure of which is incorporated herein by reference. The type of GP2 that results from cleavage of the GPI-anchor is called globular GP2 (gGP2).

The protein of SEQ ID NO: 280 is a GP2 protein and is therefore likely to be involved in regulatory membrane trafficking along the apical secretory process in various epithelial cells. Therefore, the present invention provides a protein of SEQ ID NO: 250, at least 5,8,10,12,15,20,25,30,35,40,50,60,75,100,150 of 200 of this protein. Fragments containing contiguous amino acids, or membrane sorting within the trans-Golgi network, zymogen granule membrane assembly, zymogen granule membrane inactivation during granule storage, and control of zymogen granule membrane transport at the apical plasma membrane. , Including the use of fragments with desired biological activity in modulating the release of zymogen membranes at the apical plasma membrane during exocytosis. In such embodiments, the protein of SEQ ID NO: 250, or a fragment thereof, is administered to an individual in whom it is desired to increase or decrease any of the above activities. The protein of SEQ ID NO: 250 or a fragment thereof may be administered directly to the individual, or the nucleic acid encoding the protein of SEQ ID NO: 250 or a fragment thereof may be administered to the individual. Alternatively, a formulation that increases the protein activity of SEQ ID NO: 250 may be administered to an individual. Such formulations can be identified by contacting the protein of SEQ ID NO: 250 or cells or preparations containing the protein of SEQ ID NO: 250 with a test agent and assaying whether the test agent increases the activity of the protein. For example, the test agent can be a compound or polypeptide or peptide. Alternatively, the protein activity of SEQ ID NO: 250 may be reduced by administering to the individual a formulation that interferes with such activity. A formulation that interferes with the activity of the protein of SEQ ID NO: 250 is to contact a test agent with a cell or preparation containing the protein of SEQ ID NO: 250 or the protein of SEQ ID NO: 250 and assaying whether the test agent reduces protein activity. Can be identified by For example, the formulation may be a chemical compound, polypeptide or peptide, antibody, or nucleic acid such as an antisense nucleic acid or a triple helix forming nucleic acid. In one embodiment, the invention provides for selectively identifying a tissue, preferably the pancreas and prostate, or based on the protein level of SEQ ID NO: 250 present in the sample, or where the tissue sample has more than one site of origin. In some cases, it relates to methods and compositions that use the proteins of the invention or portions thereof as marker proteins to identify them. For example, the protein of SEQ ID NO: 250 or fragments thereof can be used to raise antibodies using techniques known to those of skill in the art, including those described herein. Such tissue-specific antibodies can then identify tissue of unknown origin, such as differentiated tumor tissue that has metastasized to forensic samples, different body sites, etc., or use immunochemistry to identify different tissue types in tissue cross-sections. Can be used to distinguish. In such methods, a detectably labeled antibody is contacted with a tissue sample under conditions that promote antibody binding. The level of antibody that binds to the test sample is measured and compared to the level of binding to control cells from the pancreas or prostate or other tissues to determine if the test sample is from the pancreas or prostate.
Alternatively, the protein level of SEQ ID NO: 250 in the test sample may be measured by determining the RNA level encoding the protein of SEQ ID NO: 250 in the test sample. RNA levels can be measured using nucleic acid arrays or by methods such as in situ hybridization, Northern blot, dot blot, or other methods known to those of skill in the art. If desired, the nucleic acid sample may be subjected to an amplification reaction, such as a PCR reaction, prior to analysis. RNA levels in test samples are compared to RNA levels in control cells derived from pancreas or prostate or other tissues,
Determine if the test sample is from the pancreas or prostate.

In other embodiments, antibodies that bind to the proteins of the invention or portions thereof are used to detect, concentrate, or purify membranes or zymogen granules, using any of the techniques known to those of skill in the art. it can. For example, an antibody to the protein of SEQ ID NO: 250 or a fragment thereof can be immobilized on a solid support such as a chromatography matrix. A preparation containing the membrane or zymogen granules is contacted with the antibody under conditions that promote binding to the antibody. The membrane or zymogen granules are released from the support by washing the support and then contacting the support with an agent that dissociates the membrane or zymogen granules from the antibody. In another embodiment of the invention, the protein of SEQ ID NO: 250, or fragments thereof, can be used to diagnose disorders associated with aberrant intercellular communication or secretion. In such methods, the protein level of SEQ ID NO: 250 in the body of a patient is measured using the methods as described herein. The protein level of SEQ ID NO: 250 in patients is compared to the level in control individuals. Sequence number to compare with control individual
An increase or decrease in the 250 protein level suggests that the patient has a defect in intercellular communication or secretion. In another embodiment, the protein of SEQ ID NO: 250 or a fragment thereof is used to promote or reduce exocytosis. For example, SEQ ID
250 proteins or fragments thereof can be used to increase or decrease the release of secreted enzymes in pancreatic acinar cells or prostate cells. Thus, proteins of the invention or portions thereof, include abnormal membranes, including, but not limited to, viral or other infections, traumatic tissue damage, and genetic diseases such as pancreatitis or prostatitis, open carcinomas and lymphomas. It can be used to diagnose, treat and / or prevent transport related disorders. In such a method, SEQ ID NO: 25
0 protein, a fragment of the protein of SEQ ID NO: 250, or SEQ ID NO: 250
Formulations that increase or decrease the activity of the protein of E. coli are administered to an individual using methods as described herein. In another embodiment, the invention detects pancreatitis or prostatitis of a protein of SEQ ID NO: 250 or a fragment thereof by detecting elevated levels of protein of SEQ ID NO: 250 in a body fluid sample such as human blood, serum, urine. The method used for the diagnosis of. The protein can be detected using techniques known to those of skill in the art, including those described herein. In some embodiments, the protein of SEQ ID NO: 250 or fragments thereof can be detected by the methods described in US Pat.Nos. 5,436,169 or 5,663,315, the disclosures of each of which are incorporated by reference in their entireties. .

References US Patent Nos. Nucleic Acids Research 18 (9): 5900, (1990) Proc. Natl. Acad. Sci. USA 88 (7): 2898-2902 (1991) Proc. Natl. Acad. Sci. USA 89: 1189-1193 (1992) Eur. J. Cell Biol. Freedman et al., Annals NY Acad. Sci. Scheele et al., Pancreas 9 (2): 139-149, 1994. <protein of SEQ ID NO: 274 (internal designation number) 145-56-3-0-D5-CS)> The protein of SEQ ID NO: 274 encoded by the cDNA of SEQ ID NO: 33 is a large amount of human RNA 3′-terminal phosphate cyclase-like protein 1 ( Rcl1) (t
It is homologous to rEMBL accession number CAB89811). RNA 3′-terminal phosphate cyclase, the enzyme first identified in extracts from human HeLa cells and Xenopus oocyte nuclei, is 2 ′ of 3′-terminal phosphate group at the 3′-end of RNA. It catalyzes the ATP-dependent conversion reaction to the 3,3 'cyclic phosphodiester, resulting in activation of the 3'end of RNA molecules. A search of the database showed that the gene encoding a protein similar to human and E. coli human RNA 3'terminal phosphate cyclase is conserved among eukaryotes, bacteria and archaea, and RNA metabolism of this enzyme. Have demonstrated important functions in (Genschik P. et al.,-E
MBO J-1998, 16, p.2955-2967). Similarly, analysis of human RNA 3'terminal phosphate cyclase and related proteins from other organisms revealed that these were identified as RNA 3'terminal phosphate cyclase (Rtc) and RNA 3'terminal phosphate cyclase-like protein (Rcl). We show that it can be subdivided into two subfamilies called. These two subfamilies are almost exclusively the conserved amino acid sequences RGxxPxGGGx (where x is an amino acid, RGxx
PxGGGx shares multiple sequence elements (including hydrophobic), and despite being structurally slightly different, these two protein subfamilies have similar functions and are involved in RNA metabolism. There is. The cyclase signature is present in the proteins of the invention (positions 157-167). In addition, this protein exhibits other signatures of the RNA 3'terminal phosphate cyclase protein (pfam signature at positions 1-368, positions 12-44, and eMotif at positions 157-168).

The 3'terminal phosphate cyclases (Rtc and Rcl) are 3'-terminal in ATP-dependent reactions.
It catalyzes the conversion of'terminal phosphates to 2 ', 3' cyclic phosphodiesters, but in this reaction, nucleoside triphosphates other than ATP are cofactors with considerably poorer activity. With both of these enzymes, the cyclization of the 3'phosphate at the 3'end of RNA occurs in a three-step mechanism as follows: (a) adenylation of the enzyme with ATP; (b) the enzyme Acts on RNA-N3'P to produce RNA-N3'PP5'A; (c) Non-catalytic nucleophilic attack by a hydroxyl group adjacent at the 2'position of phosphorus in the diester bond produces a cyclic end product. The RNA 3'terminal phosphate cyclase protein is involved in RNA processing.
It has been demonstrated that multiple eukaryotic and prokaryotic RNA ligases require 2 ′, 3 ′ circular phosphate RNA ends, which means that the enzyme has multiple RNAs in both eukaryotes and prokaryotes. It is suggested that ligase is involved in the formation or maintenance of circular termini in RNA ligation substrates, which are known to be required. 2 of these ligases
Individual tRNA-splicing ligases and a prokaryotic RNA ligase of unknown function that ligates RNA ends via a variant 2 ', 5'-phosphodiester (Arn E. et al.,-RNA s
tructure and Function-Cold spring Harbor Laboratory Press-1998 p.695
-726). The involvement of these ligases in splicing nuclear tRNA precursors has been well described (Zillmann et al., --Mol Cell Biol-1991, 11, p5410-5416) (Ph
izicky E. et al.-J Biol Chem, 1992, 267, p4577-4582), but these enzymes may additionally function in the ligation reaction of virusoids and viroids (Branch A
., Science-1982, 217, p1147-1149) (Kibertis et al., -EMBO J-1985,
4, p817-827). Alternatively, cyclase may be responsible for the production of cyclic phosphate 3'ends identified in small nuclear RNA of spliceosome U6, and some other small RNAs. Furthermore, in yeast, Rcl is 18S in yeast and vertebrates.
It is associated with the U3 small nucleolar RNP (U3 snoRNP), a central element in the ribosomal RNA (rRNA) processing machinery (Billy E. et al., -EMBO J-2000, 19, p211).
5-2126). However, since Rcl does not appear to be a component of U3 snoRNP, its association with U3 snoRNP is most likely to occur in large macromolecular complexes that represent nascent ribosomes. In yeast, deficiency or inactivation of Rcl results in a defect in 18S mRNA synthesis, which reduces levels of ribosomal 40S subunits, resulting in accumulation of free 60S ribosomes and reduced polysome levels. In yeast 18S, 5.8S and 25S rRNAs are derived from the long 35S precursor. This 35S rRNA precursor is normally cleaved at the A0 site to give the 33S rRNA precursor. Then 33S rRNA
Is rapidly processed at the A1 and A2 sites to produce a 20S rRNA precursor, which is further processed into the mature 18S rRNA. Deletion or inactivation of Rcl reaches suppression of processing at the A0, A1 and A2 sites (Billy E. et al., -E
MBO J-2000, 19, p2115-2126).

The protein of SEQ ID NO: 274, or a portion thereof, is probably involved in RNA processing, as an RNA 3'terminal phosphate cyclase. Preferred polypeptides of the present invention are polypeptides containing amino acids 157-167, 1-368, 12-44, and 157-168. Other preferred polypeptides of the present invention are fragments of SEQ ID NO: 274 having any of the biological activities described herein. Assays for cyclase activity were as described in the Filipowicz article, the entire disclosure of which is incorporated herein by reference (Filipowicz W et al.,-Methods Enz.
ymol.-1990, 181, p.499-510), the Norit method, or other techniques known to those skilled in the art. Accordingly, embodiments of the present invention include the immunoprecipitation method (Billy E. et al., Described in Filipowicz's article, the protein of the present invention or a portion thereof, which is incorporated herein by reference in its entirety. -EMBO J-2000, 19, p2115-2126), or other techniques known to those of skill in the art for isolation of small nucleolar RNPs, particularly but not limited to biological samples U3 snoRNP. Compositions and methods for use in in vitro RNA engineering processing to In other embodiments, the proteins of the invention or portions thereof can be used to develop antagonists to the proteins of the invention or portions thereof to suppress or reduce cell proliferation. This indicates that the protein of the invention or a part thereof is probably involved in rRNA maturation, and thus the use of products that suppress rRNA maturation prevents the formation of functional ribosomes, which results in This may be explained by the fact that it leads to suppression of synthesis. Cells that are unable to synthesize the protein die from growth and eventually die due to the inability to regenerate the protein. One preferred embodiment of the present invention is a book for developing antagonists that is added to a sample or material as a "cocktail" in combination with other antimicrobials to stop and / or prevent the growth of unwanted contaminants. It relates to the use of the proteins of the invention or parts thereof. For example, the proteins of the invention, or portions thereof, can be used in in vitro culture to suppress unwanted bacterial and / or viral growth. In another preferred embodiment of the invention, the protein or part thereof is used for the development of an antagonist which can be administered to a patient suffering from a viral and / or bacterial infection, in particular with a virus such as HIV and HCV. be able to. This can be accomplished, for example, by targeting the antagonist to cells infected by the virus or directly to the bacteria. Once inside the cells, the antagonist suppresses or at least reduces protein synthesis, resulting in reduced bacterial and / or viral replication. In yet another preferred embodiment of the invention, the protein or part thereof is cancer, psoriasis, systemic lupus erythematosus (SLE), arthritis, endometriosis, enteropathy in immunodeficiency virus infection, venous eczema (fatty skin). Induces connective tissue sclerosis in sclerosis, causing a reduced tendency to re-epithelialize in venous ulcers), chronic irritant contact dermatitis (CICD), adult polycystic kidney disease (APKD), ichthyosis , Can be used to develop an antagonist that can be administered to a patient to suppress abnormal and / or unregulated cell growth detected in diseases such as cholesteatoma.

<Protein of SEQ ID NO: 303 (internal designation number 187-31-0-0-F12-CS) and 275 protein (internal designation number 145-59-2-0-A7-CS)> Detected in fetal kidney The 148 amino acid long protein of SEQ ID NO: 303 encoded by the cDNA of SEQ ID NO: 62, which is highly expressed in this organ, is a human RNA-associated protein HSCP250 (SPTREMBLNEW SPTREMBL SWISSPROT accession numbers AAF36170 and GEN).
It is homologous to ESEQP accession number Y84433). Potential phosphorylation sites are 32, 38, 47 (S
Amino residues), 60 (Y amino residues), 69 and 141 (T amino residues). Furthermore, this protein is the L27 ribosomal protein of Drosophila melanogaster (GENPEPT GENPEPTNEW accession number AE003576) and the 50S ribosomal L of E. coli.
It shows considerable homology with the 27 protein (SWISSPROT accession number P02427). The protein of SEQ ID NO: 303 has a putative signal peptide from amino acid positions 13-27.
Furthermore, according to the PFAM program, the proteins of the invention display the L27 ribosomal protein signature at positions 31-81. The amino acid residues at positions 64-78 are quite similar to the common pattern: GX- <LIVM> (2) -XRQRGX (5) -G, where X represents an amino acid, but any amino acid Amino acids such as
The motif in the protein of SEQ ID NO: 303 is GXIIXTQRHX (5) -G)
. Potential phosphorylation sites are 32, 38, 47 (S amino residue), 60 (Y amino residue),
It is located at positions 69 and 141 (T amino residue). One of them, the T residue at position 69, is embedded in the above-mentioned L27 ribosomal protein signature. The protein of SEQ ID NO: 275 encoded by the cDNA of SEQ ID NO: 34 is a 94 amino acid long variant of the protein of SEQ ID NO: 303. The first 81 amino acid residues of the protein of SEQ ID NO: 275 are exactly homologous to the 81 amino acid residues of the protein of SEQ ID NO: 303, but the following 13 amino acids are different. The protein of SEQ ID NO: 275 has a putative signal peptide (positions 13-27), an L27 ribosomal protein signature (positions 64-78), and a phosphorylation site (positions 32, 38, 47, 60 and 69).
In addition, candidate transmembrane segments are shown at positions 74 to 94. L27 ribosomal protein is one of the ribosomal large subunit proteins. L27 is based on sequence similarity and is based on eubacterial L27 and plant chloroplast L27.
(Encoded by the nucleus), algal chloroplast L27 and yeast mitochondrial YmL2
(MRPL2 or MRP7 gene) belonging to the family of ribosomal proteins. Of the different L27 ribosomal proteins characterized to date, E. coli is probably the most studied. The L27 protein is the smallest and most basic of the E. coli ribosome polypeptides. Techniques such as measuring protein exposure due to hot tritium irradiation have
We have shown that L27 is well exposed on the surface of the 70S ribosome of E. coli (Agafonov
Proc. Natl. Acad. Sci. 94: 12892-12897 (1997)). Chemical crosslinking and U
V-irradiation cross-linking studies have demonstrated that L27 is strongly associated with the V domain of 23S rRNA, a region containing a portion of the peptidyl transferase center (Osswald et al., Nucleic Acids Res. 18). : 6755-6760 (1990)). Direct evidence for the presence of L27 at the peptidyl transferase center was obtained through the use of a derivative of tRNAPhe containing a photoactive azidonucleotide in the 3'terminal ACCAOH sequence (Wower et al., Proc. N.
atl. Acad. Sci. 86: 5232-5236 (1989)). Analysis of a mutant Escherichia coli strain in which the rpmA gene encoding L27 was replaced with a kanamycin marker showed that L27 had an A site tRNA.
It has been suggested that it contributes to peptide bond formation by facilitating proper placement of the acceptor end of P. cerevisiae at the peptidyl transferase center (Wower et al., J. Biol.
Chem. 273: 19847-19852 (1998)). In addition, a recent study performed by Thiede and coworkers determined the precise RNA-protein contact site in the ribosomal 50S subunit of E. coli (Thiede et al., Biochem. J. 334: 39-42 (199
8)), and that Lys-71 and Lys-74 of L27 interact with U-23334 of 23S rRNA.

The proteins of SEQ ID NOs: 303 and 275 appear to be human RNA associated proteins. A preferred polypeptide of SEQ ID NO: 303 is a polypeptide comprising amino acids at positions 13-27, 64-78 and amino acid residues at positions 32, 38, 47, 60, 69 and 141. The protein of SEQ ID NO: 275 is 9 of the 148 amino acid residue protein of SEQ ID NO: 303.
It seems to be a 4-amino acid long variant. A preferred polypeptide of SEQ ID NO: 275 is
A polypeptide comprising amino acids 13-27, 64-78, 74-94, and amino acid residues 32, 38, 47, 60, and 69. Other preferred polypeptides of the invention are SEQ ID NOs: 303 or 275 having any of the biological activities described herein.
Is a fragment of One embodiment of the present invention includes the use of the proteins and nucleic acids to specifically identify cells of the kidney, particularly fetal kidney. Such cells can be detected by strong expression of the protein of the invention, and thus an antibody for detecting the expression or activity of the protein, a specific nucleic acid, or any other specifically polypeptide of the invention or Detection can be by any conventional method, including those involving detectable molecules that bind to the polynucleotide. The ability to specifically detect kidney cells is useful, for example, to determine the identity of tumor cells and to identify specific cell types and tissues, eg, for histological analysis. In another embodiment of the invention, the protein is used as a component of an in vitro eukaryotic translation system. Such systems represent a widely used protein production tool with many academic and industrial applications. Similarly, inhibitors of the proteins of the present invention, such as antibodies or dominant negatives of proteins, may abrogate the translational reaction, eg, specifically involving eukaryotic extracts, to suppress the in vitro translation system. Can be used for In another embodiment, the protein of SEQ ID NO: 303 or 275 can be used alone or in combination with other substances to bind to a nucleic acid, preferably RNA. For example, the protein of the present invention or a part thereof can be added to a sample containing RNA under conditions optimal for binding and allowed to bind to RNA. In a preferred such embodiment, the protein of the invention, or a portion thereof, is used to purify mRNA, for example to specifically isolate RNA from, for example, a particular cell type, or cells cultured under particular conditions. Can be used. Such RNAs can then be reverse transcribed and cloned and analyzed for relative expression analysis and the like. Moreover, such methods can be used to specifically remove RNA from a sample, for example during purification of DNA. In carrying out any of these methods, the protein of the invention, or a portion thereof, may be used alone or in combination with other RNA binding proteins using techniques known in the art to create affinity chromatography columns. And may be attached to a chromatographic support. The sample containing the mixture of nucleic acids to be purified is then passed through the column. Immobilizing the protein of the invention or a portion thereof on a support is particularly advantageous in embodiments in which the method is practiced on a commercial scale. This immobilization facilitates the removal of RNA from the batch of resin-bound proteins and subsequent protein reuse. Immobilization of the protein of the invention or a part thereof can be achieved according to methods known to those skilled in the art, eg by inserting any matrix binding domain into the protein. The resulting fusion product containing the protein of the invention, or a portion thereof, is then covalently or using any other means, a protein, carbohydrate or matrix (gold, "Sephadex" particles, polymer, Surface).

Yet another embodiment of the present invention relates to a method for preparing an antibody against the protein of the present invention or a part thereof. Such antibodies can be used, for example, in co-immunoprecipitation experiments to separate and purify RNA associated with the proteins of the invention.
To achieve this, antibodies to the proteins of the invention may be added to a sample containing a mixture of nucleic acids in combination with protein A or protein G sepharose beads. Immunoprecipitation conditions are known to those of skill in the art. The invention further relates to methods and compositions used to modify the proteins of the invention. In a preferred embodiment, the K amino acids of the protein of the invention are
Since these K residues appear to be important in RNA interactions, they are replaced with other base amino residues (R residues) (Thiede et al., Biochem. J. 334: 39-42 (1998)). Conversely, the R residue of the protein of the present invention may be replaced with a K residue. These substitutions are
It is predicted to change the specificity and / or affinity of the proteins of the invention for RNA molecules. Another preferred embodiment is to perform the post-translational modification of the protein of the invention significantly at the level of putative phosphorylation sites at positions 32, 38, 47, 60, 69 and 141 of SEQ ID NO: 303 described above. possible. By adding a negative charge to the proteins of the invention, these phosphorylation sites can modulate their affinity for RNA molecules. Phosphorylation of the T residue at position 69 is crucial as it is embedded in the ribosomal L27 protein signature. In other preferred embodiments, the proteins of the invention or portions thereof can be used to visualize RNA when the polypeptide binds to a suitable fusion partner or when detected by antibody search. In another embodiment of the present invention, the protein of the present invention or one thereof for specifically associating mRNA with the inner surface of the lipid bilayer of liposome and further introducing these mRNA into the cytoplasm of eukaryotic cells. Parts and compositions and methods. For example, as described above, the protein of the present invention of SEQ ID NO: 275 has 74-9 transmembrane segment candidates.
Position 4 (its carboxy-terminal end) and ribosomal L27 protein signature from 64 to
Shown at position 78. In addition, SEQ ID NO: 275 is an RNA binding protein. Preferably,
First, the specific mRNA is associated with the protein of the invention, and then the RNA / protein complex thus formed is mixed with liposomes according to methods known to those skilled in the art. These liposomes are added to in vitro cultures of eukaryotic cells. In vivo, such methods are used for the diagnosis, treatment and / or prevention of disorders associated with impaired gene transcription, such as cancer and other disorders associated with abnormal cell differentiation, proliferation or degeneration. it can.

In other embodiments, the proteins and nucleic acids can be used to modulate cell growth rates in vitro or in vivo. Studies in Drosophila show that reduced ribosomal function results in considerable inhibition of cell growth. Therefore, the compound capable of suppressing the expression or function of the protein of the present invention can be used for suppressing the growth rate of cells,
Thus, it can be used for treatment or prophylaxis in diseases or conditions associated with excessive cell growth such as, for example, cancer or inflammatory conditions. Such compounds include, but are not limited to, antibodies, antisense molecules, dominant negative proteins, and heterologous compounds that suppress protein expression or activity. <Protein of Row No. 269 (Internal Designation No. 116-115-2-0-F8-CS)> The protein of SEQ ID NO: 269 encoded by the cDNA of SEQ ID NO: 28 is mink whale ribonuclease A, which is a member of the pancreatic ribonuclease family. (Emmens M., et al., J. 157: 317-323 (1976)). Furthermore, the protein of the present invention comprises a first amino acid at position 1 to 21 and a first amino acid at position 179 to 199.
Two or two transmembrane segments are shown. The cDNA of SEQ ID NO: 28 consists of 3 exons. Exon 1 is encoded by nucleotides 1-225, exon 2 by nucleotides 226-288, and exon 3 by nucleotides 289-597. The protein of the present invention is highly expressed in the testes. Ribonucleases are proteins that catalyze the hydrolysis of phosphodiester bonds in RNA strands. Pancreatic ribonuclease is a pyrimidine-specific ribonuclease that is abundant in the pancreas of some mammalian taxa and a few reptiles. In addition to the hydrolytic function of RNA, ribonucleases have evolved to maintain a variety of other bioactivities. Such activities include antiparasitic, antibacterial, antiviral, and antitumor activities, and, in some cases, neurotoxic effects and promotion of angiogenesis. For example, bovine semen ribonuclease is antitumor (Laceetti, P. et al. (1992) Cancer Res. 52: 4582-4586), and some frog ribonucleases exhibit both antiviral and antitumor activity (Youle, RJ
(1994) Proc. Natl. Acad. Sci. USA 91: 6012-6016; Mikulski, SM et al. (1990) J. Natl. Cancer Inst. 82: 151-152; and Wu, Y. -N. Et al. , (199
3) J. Biol. Chem. 268: 10686-10693). Furthermore, angiogenin is a tRNA-specific ribonuclease that binds to actin on the surface of endothelial cells for endocytosis, translocates to the nucleus after binding, and promotes endothelial invasiveness required for angiogenesis (Moroianu, J. . and Riordan, JF (1994) Proc. Natl. Acad. Sci. USA
91: 1217-1221). Furthermore, eosinophil-derived neurotoxin (EDN) and eosinophil cation protein (ECP) are associated with neurotoxic ribonucleases (Bei
ntema, JJ et al. (1988) Biochemistry 27: 4530-4538; Ackerman, SJ (1
993) In Makino, S. and Fukuda, T., Eosinophils: Biological and Clinic
al Aspects. CRC Press, Boca Raton, Fla., pp 33-74). ECP also exhibits cytotoxic, antiparasitic, and antibacterial activity. Finally, the EDN-related ribonuclease, RNase k6, is expressed in normal human monocytes and neutrophils, suggesting a role for this ribonuclease in host defense (Rosenberg, HF a
nd Dyer, KD (1996) Nuc. Acid. Res. The protein of SEQ ID NO: 269 appears to be a ribonuclease and is therefore capable of hydrolyzing ribonucleic acid, protecting against infection and neoplasia, and neurotoxic and It is involved in several processes including angiogenesis. Preferred polypeptides of the present invention are fragments of SEQ ID NO: 269 having any of the biological activities described herein. The ribonuclease activity of a protein of the invention or a portion thereof can be assayed using any assay known to those of skill in the art, including those described in US Pat. No. 5,866,119.

In one embodiment, the subject polynucleotides and polypeptides are used to specifically detect testicular tissue and testicular-derived cells, as the protein is overexpressed in testicular tissue. For example, the protein of the present invention or a part thereof is
It can be used to synthesize specific antibodies using techniques known to those skilled in the art. Such tissue-specific antibodies can then be used to identify tissues of unknown origin, such as forensic samples or tumor tissues that have metastasized and differentiated into different body sites, etc. Can be used to distinguish between tissue types. The present invention provides methods and compositions for using the proteins of the present invention or portions thereof to hydrolyze single or multiple substrates, preferably nucleic acids, more preferably RNA, alone or in combination with other substances. Regarding For example, a protein of the invention, or a portion thereof, is added to a sample containing single or multiple substrates under conditions suitable for enzymatic activation, thus the protein catalyzes the hydrolysis reaction of the substrate (s). Exert an action. In a preferred embodiment, the protein of the invention or a portion thereof can be used alone or in combination with other nucleases to remove contaminating RNA present in a biological sample. In a further preferred embodiment, the protein of the invention, or a portion thereof, is used to remove the RNA template prior to second strand synthesis and analysis of in vitro translation products for the purpose of removing contaminating RNA from the DNA construct. use. In such an embodiment, the protein of the invention, or a portion thereof, is added to the biological sample as a "cocktail," in combination with other nucleases. The advantage of using a cocktail of hydrolytic enzymes is that a wide range of substrates can be hydrolyzed without knowing the specificity of the enzyme used or the identity of all substrates. Such nuclease cocktails are commonly used in molecular biology assays to remove unbound RNA, for example in RNase protection assays. Using a cocktail of hydrolases, a wide range of unknowns with a vast number of origins
Protect the sample from RNA contaminants. For example, the protein of the invention or a portion thereof is added to a sample where contamination of the substrate is not desired. Alternatively, the protein of the invention, or a portion thereof, may be chromatographed alone or in combination with other hydrolases using techniques known to those of skill in the art to create affinity chromatography columns for the purpose of removing substrates. It may be bound to a support. Samples containing undesired substrate are stripped of the substrate through a column. Immobilizing the protein of the invention or a portion thereof on a support is particularly convenient for those embodiments in which the method is practiced on a commercial scale. This immobilization facilitates removal of the enzyme from the product batch and subsequent reuse of the enzyme. Immobilization of the protein of the invention or a part thereof can be achieved, for example, by inserting a cellulose binding domain into the protein. Those skilled in the art will appreciate that other methods of immobilization can also be used and are described in the published literature. Alternatively, similar methods can be used to identify new substrates.

Other embodiments are described in Youle et al. (1994) supra; Mikulski et al. (1990) supra, Wu et al.
To clean or sterilize samples infected with unwanted parasites, bacteria and / or viruses using any of the assays known to those of skill in the art, including those described in (1993). Use the protein of the invention or part thereof. In a preferred embodiment, the protein is used to remove RNA virus in a sample or patient body. In another embodiment, the invention relates to compositions and methods that employ the proteins of the invention, or portions thereof, to selectively kill cells. The proteins of the invention, or portions thereof, are selected as lectins, receptors or antibodies, as described in US Pat. No. 5,955,073, the disclosure of which is incorporated herein by reference in its entirety. It binds to a recognition moiety capable of binding to cells, thereby producing a cell-specific cytotoxic reagent. In another embodiment, the protein of the invention, or a portion thereof, is used to diagnose a tumor disorder,
Used for prevention and / or treatment. In one such embodiment, the cancer of a patient can be treated or prevented by increasing the activity of the protein in the body of the patient, especially in its neoplastic or hyperplastic cells. For example, a polynucleotide encoding the protein of the present invention or a part thereof, a polynucleotide encoding the protein, or a compound that causes an increase in the activity or expression of the protein of the present invention is added to a patient, or cells derived from the patient, in vivo. Or administered in vitro. Preferably, the protein, polynucleotide, or compound is directed against neoplastic or hyperplastic cells by, for example, intratumoral injection of the molecule or by binding to a portion of the targeting moiety, such as a tumor cell-specific antibody of the molecule. Target specifically. In another embodiment, suppressing the expression or activity of the protein of the present invention in the endothelial cells of the patient, particularly the endothelial cells involved in angiogenesis, can treat or prevent cancer in the patient. Such expression or activity can be suppressed using any method, but for example, antibodies, antisense sequences, ribozymes, dominant negative forms of proteins, and small molecule inhibitors of protein activity or expression can be used.

In another embodiment, the polynucleotide and polypeptide sequences are used to diagnose cancer in a patient. In typical such embodiments, a biological sample is obtained from a patient to detect the level of the subject polypeptide or polynucleotide and compare it to a control level to determine the difference between the level observed in the patient and the control level. , Indicates the presence of cancer in the patient. In another embodiment, the protein is suppressed in mammalian cells to protect the mammalian cells from RNase-associated neurotoxicity. In typical such embodiments, protein levels are detected in the patient's cells, and elevated levels of this protein, especially in neurons, are at risk of neurotoxic effects. The level of protein expression or activity can be determined using any common method, such as an antibody, antisense molecule, ribozyme, dominant negative form of protein, or other compound capable of suppressing protein activity or expression. However, it is still suppressed. Preferably, such inhibitors are those that specifically act on mammalian neurons. <Protein of column number 390 (internal designation number 116-118-4-0-A8-CS)> The present inventors belong to the carbonic anhydrase (CA) family, more specifically, the alpha CA family, and each has a SEQ ID NO: New genes and proteins described in 149 and 390 have been provided. This novel alpha CA-related gene is found on human chromosome 1
Located in the 7q24 region. Carbonic anhydrase (EC 4.2.1.1) (CA) catalyzes the reversible hydration of carbon dioxide,
It is a zinc metalloenzyme. Nine different activated alpha carbonic anhydrases (alpha CAs) that catalyze hydration reactions have been discovered, as well as at least two alpha CA-related enzymes. The known carbonic anhydrases from the animal kingdom are all alpha CAs, also zinc enzymes, which are quite different from sequence unrelated beta and gamma CAs. The protein of SEQ ID NO: 390 was found to be highly conserved at amino acids between 20 and 59 of the protein, particularly in the multi-alignment published by Lovejoy et al., 1998 (Genomics 54, 484-493). , 45 of protein
It shows significant homology to the pfam carbonic anhydrase domain at the Gly-Ser-Glu-His motif at position -48. For localization on the chromosome, the sequence is chromosome 17 (accession number AC
002090 genbank genomic fragment) BLAST alignment,
And other genomic fragments in which the gene maps to the 17q24 region (accession number
AF064854) and alignment. The polypeptide of SEQ ID NO: 390 is a human protein 10 (Adachi, K.
And Genbank accession number A published directly in the database by Nishimori, I.
B036836), showing particularly high homology.

Human alpha CA is a highly conserved catalytic site containing a zinc coordination polyhedron that defines the active site located within a large conical cavity that extends almost to the center of the alpha CA molecule. With. One site in the cavity consists of hydrophobic hydrophobic residues with hydrophilic residues on the opposite side including Thr199 and Glu106 (see CA II enzyme). Zinc ions are located at the bottom of this gap and ionize the imidazoles of three histidine residues (His94, His96, His119 with reference to the CA II enzyme) and hydroxide ions with a pK of about 7. It forms a tetrahedral coordination bond with a water molecule called "zinc water" (Sly et al., A
nn. Rev. Biochem. 64: 375-401 (1995). Research shows that Zn-OH- / The199 / Glu106
It was revealed that networks are important for binding bicarbonate, sulfonamide inhibitors and many anion inhibitors (Liljas et al., Eur. J.
Biochem. [Good alpha CA inhibitor] It was discovered that among human alpha CA, isozymes have different tissue distribution and intracellular localization. For example, alpha CA II is expressed in the cytosol of several cell types in almost all tissues or organs, whereas
Alpha CA I is expressed on colon and erythrocytes, and alpha CA IV is on the apical surface of epithelial cells of some segments of nephron, the apical plasma membrane of the lower gastrointestinal tract, and the endothelium of some capillary beds. It is expressed on the plasma surface of the cell. SEQ ID NO: 14
The protein of SEQ ID NO: 390 encoded by the cDNA of 9 was discovered by the inventor to be expressed in the testis. Human alpha CA has been found to be involved in a variety of important biological functions, including pH regulation, CO2 and HCO3-transport, ionic transport, and water-electrolyte balance. Functions involved in alpha CA include H + secretion, HCO3-resorption, HCO3-secretion, bone resorption, and production of aqueous humor, cerebrospinal fluid, gastric acid and pancreatic juice. Of particular medical interest, it was discovered that CA II is involved in osteoporosis, and that CA II deficiency is also responsible for hereditary osteoporosis associated with renal tubular acidosis and cerebral calcification. CA activity can be measured by known means. Assays that characterize the activity of CA isozymes are described, for example, by Khalifah, J. Biol. Chem. 246: 2561-73 (1971); Chen.
Et al., Biochem 32: 7861-65 (1993); Tu et al., J. Biol. Chem. 258: 8867-8871 (198.
6); and Jewel et al., Biochem. 30: 1484-1490 (1991).

Many different CA inhibitors have been identified and several CA inhibitors have been developed as drugs. CA inhibitors are currently the main treatment for glaucoma, which reduces intraocular pressure by inhibiting CA activity and inhibiting the formation of aqueous humor ,. CA inhibitors approved for glaucoma include acetazolamide (diamox (), metazolamide (neptazan (), dorzolamide (torsopt () and brinzolamide (azopt ()). [Improved broad-acting CA inhibitor] CA inhibitors that have the ability to suppress a broad class of CA isozymes are required to suppress sympathetic activity, and partial (topical) use of CA inhibitors is advantageous over systemic use for glaucoma It has been discovered that the systemic side effects of CA inhibitors can be avoided, however, current CA inhibitors may have limited efficacy with respect to their ability to completely inhibit all CA activity. The law is dorzolamide (torsopt (
) Is an inhibitor of CA II, but can inhibit CA VI only slightly (Hoyng
Et al .. Drugs, 50 (3): 411-434 (2000). Inhibitors of CA II are believed to be unable to suppress CA I or other CAs, and thus reduce potency as other CAs compensate for the reduced CA II activity (Sly and Hu, Ann. . Rev. Biochem.
64: 375-401 (1995)). In one example, human erythrocytes have 5-6 times more CA I than CA II, but their activity is only about 15% that of CA II. Therefore, CA I
Contributes to approximately 50% of CA activity (Dodgon et al., J. Appl. Physiol. 64: 1492-8.
0 (1988). In addition, CA I is believed to have different inhibitor susceptibility properties than CA II, as CA I exhibits less sensitivity to, for example, sulfonamide inhibitors. Conversely, CA II and CA IV are much more resistant to the inhibitory effect of halogen ions than CA I. (Sly et al., (1995), supra).
Thus, the presence of significant amounts of CA residual activity in cells or tissues of interest may be due to other CAs, including the polypeptide of SEQ ID NO: 390. Thus, in one aspect, alpha CA-related nucleic acids and polypeptides may be useful in identifying compounds capable of inhibiting the reversible hydration of alpha CA catalysts.
In one aspect, a method is implemented for identifying or selecting a CA inhibitor capable of suppressing the activity of the polypeptide of SEQ ID NO: 390. In other embodiments, this method is performed to identify or select CA inhibitors that are capable of broadly suppressing the activity of multiple CA enzymes. Nucleic acid and polypeptide sequences of the invention are used for computer-based drug design, or to make predictions of CA inhibitor candidate binding, given the extensive structural information about publicly available CA enzymes. be able to. In a preferred embodiment, the nucleic acids and polypeptides of the invention are used in drug screening assays. The assay may be a cell-based or a non-cell-based assay. In one embodiment, a nucleotide or polypeptide sequence of the invention is contacted with a candidate CA inhibitor (such as a CA II inhibitor) to bind the inhibitor candidate to a polypeptide of the invention or to activate a polypeptide of the invention. To detect. The activity of the polypeptide of the present invention may be CA activity, or any other suitable activity possessed by the polypeptide of the present invention that is suppressed by the binding of a candidate substance. Assays to detect hydration of carbon dioxide are known,
Referenced above is a reference. In a preferred embodiment, a panel of CA isozymes comprising a polypeptide of the invention is provided with CA I, CA III, CA IV, CA VI and CA-
Screen for candidates containing one or more enzymes selected from the group consisting of CA-RP, including but not limited to RP VII, CA-RP X and CA-RP XI. In a preferred embodiment, CA inhibitor candidates are selected based on their ability to broadly suppress CA isozymes capable of catalyzing the hydration of carbon dioxide. Methods of performing such drug screening assays are known in the art. In one embodiment, the methods further described herein, for example, International Patent Application Publication No. WO 00/58510, the disclosure of which is incorporated by reference in its entirety herein, particularly “ Methods for screening substances interacting wi
Test the binding of the drug using the method described in the section entitled "th ... Drug binding assays in a large panel of isozymes may be performed in high throughput format using commercially available binding assay systems (Graffinity Pharmaceutical Design GmbH, Heidelberg, Germany).

The method according to the invention generally comprises pH control, Co2 and HCO3-transport, ion transport,
And can be used to identify or select candidate compounds for the treatment of disorders characterized by disorders in water and electrolyte balance. Improved Selective CA Inhibitors In other therapeutic approaches, CA inhibitors are delivered orally. However,
Systemic delivery can affect CA enzymes present in other tissues or organs and can have adverse side effects. CA II inhibitors may be associated with other CA isozymes such as CA I or CA-related proteins such as CA-RP VIII, X, XI or CA RPTP-beta (CA-R
P) (Tashian et al., In "Carbonic Anhydrase: New Horizons", WR Chegwidden,
ND Carter and YH Edwards, Eds., Birkhauser, Basel); Adachi, K et al., Ge
nbank accession number AB036836; Lovejoy et al. (1998); Peles et al. (1995)) or is expected to partially or completely suppress the CA polypeptide of SEQ ID NO: 390. For example, CA-RPTP beta has a CA region that is inactivated or reduced in CA activity, but is considered to be involved in ligand binding or participation in protein complexes, considering the binding of contactin by the extracellular region. To be (Peles et al., Cell 82:25.
1-260 (1995); Tashian et al., (1998), supra). Inhibition of isozymes such as CA-RPTP beta, or isozymes of the invention by systemic treatment with non-selective drugs can cause adverse side effects. In one example, an oral CA inhibitor for the treatment of glaucoma (eg acetazolamide) is
It is effective with no side effects on the eyes, and at the same time has paresthesias, fatigue, depression,
He showed significant systemic effects, including renal stones and gastrointestinal illnesses such as nausea and diarrhea. Prevention of side effects is especially important because CA inhibitors (Hoyng et al., 2000, supra) are commonly used permanently (eg, for glaucoma) or for extended periods of time. Therefore, selective CA inhibitors that have a considerable ability to suppress the CA isozyme of interest over other CA isozymes may therefore provide an improved means of treating disease. In one embodiment, the nucleotide and polypeptide sequences of the invention can be used in the design of selective CA inhibitors. Studies have revealed that alpha CA has different inhibitor binding properties (Sly et al., (1995
), Supra), suggesting that it is possible to provide compounds that suppress the CA isozyme of interest, such as CA II, without binding or suppressing related enzymes such as the polypeptide of SEQ ID NO: 390. . Nucleic acid and polypeptide sequences of the invention can be used for computer-based drug design, or prediction of CA inhibitor candidate binding, given the widespread structural information about commonly available CA enzymes. In a preferred embodiment, the nucleic acids and polypeptides of the invention are used in drug screening assays, including both cell-based and non-cell based assays. In one embodiment, a nucleotide or polypeptide sequence of the invention is contacted with a candidate CA inhibitor (such as a CA II inhibitor) to detect binding of the inhibitor candidate to a polypeptide of the invention or activity of a polypeptide of the invention. To do. The activity of the polypeptide of the present invention may be CA activity or an appropriate activity other than CA activity possessed by the polypeptide of the present invention that is suppressed by the binding of a candidate substance. In a preferred embodiment, a panel of CA isozymes comprising a polypeptide of the invention is prepared by combining the polypeptide of SEQ ID NO: 39 with CA I, CA III, CA IV, C
A VI and CARP including but not limited to CARP VII, CARP X and CARP XI
Screening for candidate substances containing one or more enzymes selected from the group consisting of: In a preferred embodiment, CA inhibitor candidates are directed against one or more non-catalytic CA-related proteins in order to eliminate undesired suppression of these enzymes, which may be involved in other important physiological functions. To screen. Methods for performing such drug screening assays are known in the art.

[Increase in alpha CA activity for treatment of alpha CA deficiency] Further, the polypeptide of the present invention can be used as a source of CA activity such as for the purpose of treating disease. Carbonic anhydrase deficiency is the cause of multiple illnesses, including osteopetrosis (abnormally dense bone) renal tubular acidosis, cerebral calcification and mental retardation. Furthermore, carbonic anhydrase-related proteins have been described to be associated with cone-rod retinal dystrophy (Bellinghan et al., 1998, Biochem. Biophys. Res. Com.
m .: 253, 364-367). Thus, in one aspect, the invention comprises increasing CA activity by providing increased activity of the polypeptide of SEQ ID NO: 390. The increased activity of the polypeptide of SEQ ID NO: 390 can be provided by suitable means, as further described herein. For example, the activity may be as determined by the SEQ ID NO:
A vector containing the nucleotide sequence of 149 is introduced, the cell is processed with a compound capable of increasing the expression of the polypeptide of the invention, and / or the cell or patient is directly treated with the polypeptide of SEQ ID NO: 390. It can be provided by processing. In a preferred embodiment, the polypeptide of the invention comprises at least one amino acid substitution, deletion or insertion. In one aspect, such amino acid changes are preferably carried out at the catalytic site; preferably said amino acid changes involve the substitution, deletion or insertion of His residues, preferably said amino acid changes are according to the invention. It increases the CO2 hydration activity of the polypeptide. [Metal Ion Biosensor] In a further embodiment, a metal ion biosensor can be designed based on the polypeptide of SEQ ID NO: 390. Determination of metal ion concentration in serum, cytoplasm and complex media such as seawater is an important analytical function requiring high sensitivity and selectivity. Biosensors may be particularly useful for detecting intracellular and intercellular fluxes of metal ions. Such biosensors can take advantage of the metal binding capacity of the polypeptides of the invention, as described by Thompson et al., Who developed such biosensors based on the CA enzyme (CA II). Biosensors are useful for detecting metal ion flux, for example in the central nervous system. Zinc-containing neurons throughout the mammalian cerebral cortex, striatum and amygdala have been shown to depolarize and calcium-dependently release their zinc in vitro and in vivo. This zinc release was suggested to act as a transsynaptic neuromodulator, which was further linked to excitotoxic neuronal cell death. A CA-based biosensor, developed by Thomspon et al., Clarified that zinc is present in and can be detected in the extracellular fluid of neurons. (Thompson et al., J. Neurosci Methods
96: 35-45 (2000)). CA-based biosensors have been shown to be highly selective, detecting sub-picomolar levels of Cu, which can be achieved by mass spectrometry. It is sensitivity. Sensors based on CA II isozymes have been shown to detect picomolar levels of Zn and Cu, as well as nanomolar levels of Cd, Co and Ni.

(Thompson et al., Anal. Biochem. 267: 185-195 (1999)). Furthermore, CA-based biosensors also showed selectivity for potential interferents in biological systems, such as Mg and Ca, present at the mM level in extracellular fluids. (Thompson et al., (2000),
(See above). The polypeptide-based biosensor of the present invention is based on the high selectivity and sensitivity of the CA isozyme for zinc. Since binding of the Zn to the active site of the enzyme affects the ability of the enzyme to bind the CA inhibitor, a CA inhibitor that exhibits a detectable change when bound to the polypeptide of the invention is used to inhibit the inhibitor, Therefore, it is possible to detect the amount of the polypeptide bound to Zn. Next, the amount of the polypeptide bound to Zn is determined by the concentration of free Zn and the polypeptide of the present invention and the dissociation constant of zinc. In one example, the binding of the CA inhibitor to the polypeptide of the invention is detected using a fluorescent inhibitor, which upon binding the polypeptide of the invention, exhibits a detectable change in the fluorescence emission wavelength of polarized light. . In one example, it is a fluorescent substance
A fluorescent sulfonamide such as ABD-N is used (Thompson et al., (2000), supra). Artificial CA Enzymes CA isozymes have been shown to have different levels of catalytic activity and efficiency. In a preferred embodiment, the polypeptides of the present invention are CO2 hydrated and / or CO2 hydrated / Alternatively, it can be modified to increase zinc binding. In particular, studies have been conducted to characterize the residues that are important in maximizing CA activity, allowing the design of CA isozymes with desired levels of activity. Essential components of zinc binding, CO2 hydration activity and stability in CA isozymes are described in Lindskog, Pharmacol. Ther. 74 (1): 1-20 (1997) and Sly (1995), supra. Has been done. In one example, a CA III study was to convert Phe198 residues to Leu198 residues (
The result showed that the activity increased 25-fold when it was changed to CAII). (Chen et al., (1993
), Supra). CA II catalysis was also greatly enhanced by substituting the Thr200 residue with His, similar to the normal CA I enzyme. Most strikingly, the CA-related protein (CA-RP), in its native form, lacks critical residues at the catalytic site and has no detectable CO2 hydration activity, Only two point mutations resulted in active CA. (Sjoblom et al., FEBS Lett. 398: 322-325 (1996)).

Thus, in the embodiment using the polypeptide of the present invention, the source of CO2 hydration, or the zinc binding property, introduces at least one amino acid substitution, deletion or insertion into the polypeptide of the present invention. It is advantageous to modify by In one aspect, such amino acid changes are preferably carried out at the catalytic site; preferably said amino acid changes involve the substitution, deletion or insertion of His residues, preferably said amino acid changes are according to the invention. It increases the CO2 hydration activity of the polypeptide. Optimal amino acid changes will be determined by one of skill in the art, especially in light of sequence comparisons that can be made using the many well characterized CA isozymes. <Protein of SEQ ID NO: 252 (Internal Designation No. 105-089-3-0-G10-CS)> The protein of SEQ ID NO: 252 is encoded by the cDNA of SEQ ID NO: 11.
Accordingly, all features and uses of the polypeptide of SEQ ID NO: 252 described elsewhere in this application also relate to the polypeptide encoded by the human cDNA which is clone 105-089-3-0-G10-CS. It will be understood. Moreover, all features and uses of the nucleic acid of SEQ ID NO: 11 described elsewhere in this application also relate to the nucleic acid encoded by the human cDNA which is clone 105-089-3-0-G10-CS. Will be understood. It is present in excess in the fetal brain. The protein of SEQ ID NO: 252 encoded by the cDNA of SEQ ID NO: 11 is mainly distributed in the prostate and salivary glands. The protein of SEQ ID NO: 252 is represented by International Patent Application Publication WO9827205-A2 (explaining the protein isolated from a human adult salivary gland cDNA library), International Patent Application Publication WO9839446-A2, International Patent Application Publication WO983944.
It is homologous to the sequence described in 6-A2. The entire disclosure of each of the aforementioned international patent application publications is incorporated herein by reference. The protein of SEQ ID NO: 252 is also homologous to the polypeptide described in International Patent Application Publication WO9835229-A1, the disclosure of which is incorporated herein by reference in its entirety. Wo9835229-A1 contains a portion of the protein of SEQ ID NO: 252 (amino acid 2
A peptide of 27 amino acid residues corresponding to 23/27 of 0-46) is described. This corresponds to the preservation of 3 out of 4 changes, showing an identity of 85%, resulting in a homology of 96%. The protein described in WO 9835229 was identified in reflex tears collected from 12 non-contact lens-worn women. The nasal mucosa was lightly rubbed with a cotton swab to stimulate reflex tears. Two different batches were collected from two different groups and examined by analytical and preparative second dimension electrophoresis. After separation in the second dimension and transfer to a PVDF membrane, spots of the identified protein (by 0.1% (w / v) Coomassie blue) were loaded onto a Hewlett-Packard cartridge compatible with the membrane. Sequencing was performed on a G1005A (Hewlett Packard, CA) sequenator. It was revealed that one of the identified proteins migrates to 25 kDa and has five isoforms with different pIs. Two of these were N-terminally sequenced to obtain sequences for the above peptides with pIs of 5.0 and 4.4. The different isoforms indicate that this protein is post-translationally modified, including sialylation or acylation. Different degrees of presence of these isoforms may reflect the pathology of the individual. Accordingly, one embodiment of the present invention is directed to a protein of SEQ ID NO: 252 or SEQ ID NO: 252 in an individual.
The detection or diagnosis of a disease by determining the activity or level of the polynucleotide encoding the protein of For example, the secreted protein of SEQ ID NO: 252 in an individual can be detected non-invasively by measuring the protein level in body fluids that secrete that protein, such as tears and saliva. Such methods can be used in both humans and animals. After cleavage of the signal peptide, the protein of SEQ ID NO: 252 may be secreted into tears and possibly body fluids including saliva.

Furthermore, the protein of SEQ ID NO: 252 can be used for the screening of non-ocular diseases, especially by analyzing tears for marker proteins indicating cancer and genetic diseases. Furthermore, the altered chromatographic profile of the isoform of the protein of SEQ ID NO: 252 (eg two-dimensional gel) is indicative of the pathology of the individual.
For example, one can determine whether an individual is afflicted with a disease by measuring the level of a marker protein related to the protein of SEQ ID NO: 252. Alternatively, tears can be analyzed for levels of different isoforms of the protein of SEQ ID NO: 252 to determine if the pattern of such isoforms is indicative of disease. The protein of SEQ ID NO: 252 or fragments thereof can be used as an eye lubricant or detergent. This protein may be included in contact lens washes and preservatives. In addition, this protein is used in daily redness or surgery /
It may be useful as a component of eye wash fluids (eg eye drops) used for healing after laser intervention. For example, proteins can be used to reduce eye inflammation. Alternatively, the antibacterial properties can be exploited by including the protein of SEQ ID NO: 252 or fragments thereof in ophthalmic solutions, creams or ointments, as well as common topical creams or ointments. Accordingly, the invention provides a protein of SEQ ID NO: 252, at least 5,8,10,12,15.
, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150 or 200 contiguous amino acid fragments, or in an individual, for treating or ameliorating a disease,
Including uses of fragments having the desired biological activity. In such an embodiment,
The protein of SEQ ID NO: 252, or a fragment thereof, is administered to an individual in whom it is desired to increase or decrease any of the activities of the protein of SEQ ID NO: 252. The protein of SEQ ID NO: 252 or fragment thereof may be administered directly to an individual, or the nucleic acid encoding the protein of SEQ ID NO: 252 or fragment thereof may be administered to an individual. Alternatively, a formulation that increases the protein activity of SEQ ID NO: 252 may be administered to the individual. Such formulations can be identified by contacting cells or preparations containing the protein of SEQ ID NO: 252 or the protein of SEQ ID NO: 252 with a test agent and assaying whether the test agent increases the activity of the protein. For example, the test agent can be a chemical compound or polypeptide or peptide.

Alternatively, the protein activity of SEQ ID NO: 252 may be reduced by administering to the individual a formulation that interferes with such activity. Formulations that interfere with the activity of the protein of SEQ ID NO: 252 contact a cell or preparation containing the protein of SEQ ID NO: 252 or the protein of SEQ ID NO: 252 with a test agent and determine whether the test agent reduces protein activity. It can be identified by For example, the formulation can be a compound, polypeptide or peptide, antibody, or nucleic acid such as an antisense nucleic acid or a triple helix forming nucleic acid. In one embodiment, the invention selectively identifies the origin of a sample, such as saliva or tears, based on the protein level of SEQ ID NO: 252 in the sample, 2
Methods and compositions that employ the proteins of the invention or portions thereof as marker proteins to identify sources from which one or more samples may be derived.
For example, the protein of SEQ ID NO: 252 or fragments thereof can be used to raise antibodies using techniques known to those of skill in the art, including those described herein. Such tissue-specific antibodies can then identify tissues of unknown origin, such as tumor tissues that have metastasized and differentiated into forensic samples, different body sites, etc., or immunochemistry can be used to identify different tissue types in tissue cross sections. Can be used to distinguish. In such methods, the sample is contacted with a detectably labeled antibody under conditions that promote antibody binding. The level of antibody binding to the test sample is measured and compared to the level of binding to control cells from saliva or tears, or tissues other than saliva or tears,
Determine whether the test sample is from saliva or tears. Alternatively, the protein level of SEQ ID NO: 252 in the test sample may be measured by determining the RNA level encoding the protein of SEQ ID NO: 252 in the test sample. RNA levels can be measured using methods such as nucleic acid arrays or in situ hybridization, Northern blots, dot blots, or other methods known to those of skill in the art. If desired, the nucleic acid sample may be subjected to an amplification reaction, such as a PCR reaction, prior to analysis. The RNA level in the test sample is compared to the RNA level in control cells from saliva or tears or tissues other than saliva or tears to determine whether the test sample is from saliva or tears. In other embodiments, antibodies that bind to the proteins of the invention or portions thereof are used for the detection, enrichment, or purification of cells expressing the protein of SEQ ID NO: 252, including techniques known to those of skill in the art. it can. For example, an antibody against the protein of SEQ ID NO: 252 or fragments thereof can be immobilized on a solid support such as a chromatography matrix. A preparation comprising cells expressing the protein of SEQ ID NO: 252 is contacted with the antibody under conditions that promote binding to the antibody. The cells are released from the support by washing the support and then contacting the support with a drug that causes the cells to dissociate from the antibody.

In another embodiment of the invention, the protein of SEQ ID NO: 252 or fragments thereof can be used to diagnose disorders associated with altered expression of the protein of SEQ ID NO: 252. In such methods, the protein level of SEQ ID NO: 252 in diseased individuals is measured using methods such as those described herein. The protein level of SEQ ID NO: 252 in a diseased individual is compared to the level in a healthy individual to determine if the individual has a protein level of SEQ ID NO: 252 indicative of disease. <Protein of SEQ ID NO: 308 (Internal Designation No. 187-41-0-0-i21-CS)> The protein of SEQ ID NO: 308 is encoded by the cDNA of SEQ ID NO: 67.
Accordingly, all features and uses of the polypeptide of SEQ ID NO: 308 described throughout this application also relate to the polypeptide encoded by the human cDNA which is clone 187-41-0-0-i21-CS. It will be understood. Moreover, all features and uses of the nucleic acid of SEQ ID NO: 67 described elsewhere in this application also relate to the nucleic acid encoded by the human cDNA which is clone 187-41-0-0-i21-CS. Will be understood. The protein of SEQ ID NO: 308 is the nf87_1 human secretory protein of International Patent Application Publication WO 9935252-A2 (the publication of which is incorporated by reference in its entirety), International Patent Application Publication WO 9906548-A2. Amino acid 26 of the human secretory protein of SEQ ID NO: 441 (the disclosure of which is incorporated herein by reference in its entirety).
~ 129, and its publication is highly homologous to amino acids 26-114 of the human secretory protein of SEQ ID NO: 439 of International Patent Application Publication WO 9906548-A2, the entire text of which is incorporated herein by reference. . Therefore, the protein of the present invention seems to be a polymorphic variant of nf87_1. Since most of the proteins with high homology to the sequences of the present invention have longer 5'ends, it is possible that the proteins of the present invention are truncated / splice variants of these proteins. The protein of SEQ ID NO: 308 was identified from among cDNAs in a library prepared from brain. Analysis of tissue distribution by database analysis using BLAST,
It indicates that the mRNA encoding this protein was detected mainly in kidney, liver, and cancer prostate.

The protein of SEQ ID NO: 308 is the human interferon inducible (IFI) protein isoform p27 (63%), HIFI (50% identity), and the interferon-inducible protein 6-16 precursor (IFI-6-). 16 and 36%) show chemical and structural homology. Furthermore, the protein of the present invention is a human erythropoietin (EPO) primary response gene, the publication of which is incorporated by reference in its entirety into the present specification, International Patent Application Publication WO 9906063-A2, EPRG3pt shows structural homology (40% identity). The present invention therefore relates to the nucleic acid and amino acid sequences of the novel IFI proteins and the use of these sequences in the diagnosis, research, prevention and treatment of diseases. The protein of SEQ ID NO: 308 contains 105 amino acids. Amino acid alignments and hydrophobicity plots have a predicted signal peptide sequence spanning 31-43 and two predicted transmembrane domains spanning residues 17-37 and 48-68. Thus, one embodiment of the invention is a polypeptide that includes a signal peptide and / or one or more transmembrane domains. Interferons (IFNs) are part of a group of intercellular messenger proteins known as cytokines. α-IFN is the product of a multigene family of at least 16 members, whereas b-IFN is the product of a single gene. Furthermore, α- and β-IFN are known as type I IFN. Type I IFN is produced in various cell types. Type I IFN biosynthesis is stimulated by viruses and other pathogens, and diverse cytokines and growth factors. Γ-IFN, also known as type II IFN, is produced in T cells and natural killer cells. The antigen used to sensitize the organism stimulates type II IFN biosynthesis. Both α- and γ-IFN are immune modulators and anti-inflammatory agents, macrophages,
Activates T cells and natural killer cells. IFN is part of the body's natural defenses against viruses and tumors. IFN exerts these defenses by affecting the functioning of the immune system and by direct action on pathogens and tumor cells. IFNs mediate these multiple effects, in part by inducing the synthesis of many cellular proteins. Some interferon inducible (IFI) genes are similarly induced by either byα-, β- and γ-IFN. Other IFI genes are preferentially induced by type I or type II IFN. The diverse proteins produced by the IFI gene have antitumor, antiviral and immunomodulatory functions. Tumor antigen expression in cancer cells is increased by α-IFN, making cancer cells more susceptible to immune rejection. IFI proteins synthesized in response to viral infection are known to suppress viral functions such as cell penetration, decoating, RNA and protein synthesis, assembly and release (Hardman JG et al. 25 (1996) The Pharmacological Basis. of Therapeuti
cs, McGraw-Hill, New York NY pp 1211-1214, the publication of which is incorporated herein by reference in its entirety). Type II IFN is a major histocompatibility complex (MHC
) It stimulates the expression of proteins and is therefore used to enhance the immune response.

The IFI gene, known as 6-16, encodes an mRNA that is highly induced by type I IFN in various human cells (Kelly JM et al. (1986) EMBO J 5:16.
01-1606, the entire text of which is incorporated herein by reference). After induction, 6-16 mRNA accounts for 0.1% of total cellular mRNA. 6-16 mRNA is only present at very low levels in the absence of type I IFN and is slightly induced by type II IFN. 6-16
mRNA encodes a 130 amino acid hydrophobic protein. The first 20-23 amino acids contain a putative signal peptide. The 6-16 protein has at least two predicted transmembrane regions ending in a negatively charged C-terminus. The p27 gene encodes a protein that exhibits 41% amino acid sequence identity to the 6-16 protein. The p27 gene is expressed in several breast tumor cell lines and gastric cancer cell lines. In other breast tumor cell lines, HeLa cervical cancer cell lines, and fetal lung fibroblasts, p27 expression occurs solely by α-IFN induction. In one breast tumor cell line, p27 is separately induced by estradiol and IFN (Rasmussen UB et al. (1993) Cancer Res 53: 4096-4101, the publication of which is incorporated herein by reference in its entirety. ing). Expression of p27 was analyzed in 21 primary invasive breast cancers, 1 breast cancer bone metastasis, and 3 breast fibroadenoma. High levels of p27 were detected in about half the primary carcinoma and bone metastases but not in fibroadenoma. These observations suggest that some breast tumors, compared to other breast tumors,
It has been suggested to produce high levels of type I IFN or have increased susceptibility to type I IFN (Rasmussen UB et al., Supra). Furthermore, the p27 gene is expressed at significant levels in normal tissues including colon, stomach and lung, but not in placenta, kidney, liver or skin. (Rasmussen UB et al., Supra). Small products of the hydrophobic IFI gene may contribute to viral resistance. The hepatitis C virus (HCV) -inducible gene, 130-51, was isolated from a cDNA library prepared from chimpanzee liver during the acute phase of infection. The protein product of this gene is
97% identity to human 6-16 protein (Kato T et al. (1992) Virology 190: 85
6-860, the full text of which is incorporated herein by reference). The authors of the aforementioned article suggest that HCV infection behaviorally induces IFN expression, which in turn induces expression of the IFI gene, including 130-51. IFI proteins synthesized in response to viral infection are known to suppress viral functions such as penetration, decoating, RNA or protein synthesis, assembly or release. The 130-51 protein may suppress one or more of these functions in HCV. I
The FI protein can suppress multiple functions of a particular virus.
Furthermore, the major suppressive effects on IFI proteins differ among virus families (Hardman JG, supra, p1211, the publication of which is incorporated herein by reference).

The HIFI protein (International Patent Application Publication No. WO 9812223-A2, the publication of which is incorporated herein by reference in its entirety), was identified from among the cDNAs of a library made from human neonatal kidney. It is a human sequence. LIFESEQTM database (Inc
yte Pharmaceuticas, Palo Alto, CA) Northern blot analysis using HIFI
It shows that mRNA was detected only in the neonatal kidney. HIFI protein is 104
Consists of amino acids of p27, 6-16 and 130-51, 55%, 45%, and 4 respectively.
It shows 6% amino acid sequence identity. Based on the chemical and structural homology between the protein of SEQ ID NO: 308 and the hydrophobic IFI small proteins of human and chimpanzee, the protein of SEQ ID NO: 308
It is considered to be synthesized when interferon is produced in infection, inflammation, autoimmune disease, etc. Interferon can cause viral infections, inflammation, autoimmune diseases,
And in cancer as a response to diverse cytokines and growth factors. Thus, the protein of SEQ ID NO: 308 or a fragment thereof can be used in the diagnosis and treatment of diseases, including but not limited to: rheumatoid arthritis, Graves' disease, systemic lupus erythematosus, autoimmune hepatitis. , Wegener's granulomatosis, sarcoidosis, polyarthritis, pemphigus, pemphigoid, erythema multiforme, Sjogren's syndrome, inflammatory bowel disease, multiple sclerosis,
Autoimmune disorders such as myasthenia gravis, scleritis, type I diabetes, insulin-dependent diabetes mellitus, lupus nephritis, and allergic encephalomyelitis; leukemia, lymphoma (
Hodgkin and non-Hodgkin), sarcomas, melanomas, adenomas, solid tissue carcinomas, hypoxic tumors, squamous cell carcinomas of the mouth, pharynx, larynx, and lungs, urogenital cancers such as cervical and bladder cancers, hematopoietic cancers, heads Proliferative disorders including various forms of cancer such as head and neck cancer, and nervous system cancers, papillomas, atherosclerosis, benign lesions such as angiogenesis, viral infections, especially HCV and HIV infections, and other pathogenesis Infection (eg Leishmania)
. In addition, the protein of SEQ ID NO: 308 or fragments thereof can be used to treat diseases associated with inflammation or immune dysfunction (eg rheumatism and osteoarthritis and AIDS). Another embodiment of the invention relates to the use of the protein of SEQ ID NO: 308 or fragments thereof to treat and / or prevent the disease effects of bacterial infection in pregnant mammals, such as spontaneous abortion and maternal death. In a preferred embodiment, the proteins of the invention can be used to counteract the effects of bacterial endotoxin lipopolysaccharide (LPS). Methods for using such compositions are described in Dziegielewska and Andersen, Biol. Neonate, 74:
372-5 (1998), the disclosure of which is incorporated herein in its entirety by reference.

Furthermore, the protein of SEQ ID NO: 308 or a fragment thereof is useful as a reagent for analyzing the regulation of gene expression by interferons and other cytokines in both normal and diseased cells. The protein of SEQ ID NO: 308 or fragments thereof can be used to identify specific molecules such as agonists, antagonists or inhibitors that bind to it. Another embodiment of the invention is the protein of SEQ ID NO: 308 for identifying and / or quantifying interferon family cytokines and other cytokines such as IL10 and tumor antigens that may interact with the proteins of the invention. Or regarding the usage of the fragment. In addition, the protein of SEQ ID NO: 308 or a fragment thereof can be included in a pharmaceutical preparation for the treatment of cancer or the prevention / treatment of other diseases associated with altered expression of the proteins of the invention. In another embodiment of the invention, the protein of SEQ ID NO: 308 or a fragment thereof prevents the binding of endogenous cytokines to their natural receptors, which results in regulatory signals generated by cell proliferation or ligand receptor binding events. Can be used to suppress and / or modulate the effects of cytokines and Il-2, TNFalpha, CTLA4, CD28, and other related molecules. The protein of SEQ ID NO: 308 or a fragment thereof can be injected into the affected tissue either intraperitoneally, intravenously, subcutaneously or directly to affect disease, such as autoimmune, inflammation and tumor models. Useful for correcting defects in in vivo models. DNA encoding the protein of SEQ ID NO: 308 or fragments thereof is useful in diagnostic assays for diseases / disorders associated with expression of the proteins of the invention. Diagnostic assays are useful for identifying the absence, presence, and overexpression of the proteins of the invention, as well as monitoring regulatory levels of the proteins of the invention during therapeutic intervention. further
The DNA is introduced into an effective eukaryotic expression vector for gene therapy of the above diseases including tumors and / or correction of any of the above proteins including the protein of the present invention for pathological or genetically induced defects. It can also be directly targeted to a specific tissue, organ, or cell population for use. In addition, DNA may be used to design antisense sequences and ribozymes that can be administered to alter gene expression in tumors and pathogen-infected cells, or to influence cytokine and growth factor expression. In vivo delivery of genetic constructs into a subject can affect expression of the proteins of the invention or expression and / or activity of other proteins such as cytokines, growth factors, their receptors and / or tumor antigens. Can be developed to the extent that it targets a specific cell type, such as a tumor that is In addition, studies on the detection of unknown upstream sequences (eg, promoters and regulators) using conventional methods, and the regulation of gene expression by interferons and other cytokines, growth in normal and diseased cells, and transcription factors Is also useful. Hybridization probes are useful for detecting DNA encoding proteins (or related molecules) of the invention in biological samples and for mapping naturally occurring genomic sequences to specific chromosomes / chromosomal regions. Use of this DNA to produce and / or treat in vivo animal models of disease, including infection, inflammation, susceptibility or resistance to tumors and autoimmune diseases, and tumor therapy based on vaccine, knockout and transgene technology. You can

Antibodies to the protein of SEQ ID NO: 308, or fragments thereof, are useful in diagnosing conditions and diseases associated with its expression, and quantifying the proteins of the invention (eg, assays that monitor patients during therapeutic intervention). is there. Antibodies specific for the protein may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments produced by the Fab expression library. Neutralizing antibodies are particularly preferred for diagnostics and therapeutics. Diagnostic assays for proteins of the present invention include methods of detecting proteins of the present invention in human body fluids or extracts of cells or tissues using antibodies and labels. The proteins of the invention and their catalytic or immunogenic fragments, or oligopeptides thereof, can be used to screen therapeutic compounds in any drug screening technique, including high throughput. Methods that can be used to quantify the expression of nucleotides or proteins of the invention include polymerase chain reaction (PCR), RT-PCR, RNAse protection, Northern and Western blots, enzyme linked immunosorbent assay (ELISA), radioimmunoassay. Assays (RIA), Fluorescence Activated Cell Sorting (FACS), Immunoprecipitation, and Chromatography include, but are not limited to. Iron-restricted erythrocytes are clearly formed under conditions of massive blood loss, EPO treatment, or both. However, there are considerable disadvantages to iron therapy intravenously or orally. In addition, traditional biochemical markers used for iron stores in patients with anemia of chronic illness help assess iron status (Lawrence T
(2000) Blood 96: 823-833, the entire disclosure of which is incorporated herein by reference. The protein of SEQ ID NO: 308 is human erythropoietin (EPO).
Since it shows homology to EPRG3pt, which is the primary response gene, it promotes erythropoiesis,
It can be used to more safely monitor the levels of intravenous iron preparations in patients with blood loss anemia, especially those receiving EPO treatment. The hydrophobic IFI protein of SEQ ID NO: 308, or a fragment thereof, can be used to diagnose pathologies associated with its induction. For example, the protein of SEQ ID NO: 308 or a fragment thereof is useful in diagnosing and treating conditions associated with tumors, viral infections, inflammation, or impaired immunity, chronic blood loss or anemia of chronic disease, hemochromatosis, and EPO treatment. Can be. Furthermore, this protein
It can be used to study the regulation of gene expression in normal and diseased cells by IFNs and other cytokines, and hormones and growth factors.

The protein of SEQ ID NO: 308 or fragment thereof can be injected into the affected tissue by intraperitoneal, intravenous, subcutaneous or direct methods to induce autoimmunity, inflammation, anemia, iron overload and It is useful for correcting defects in in vivo models of disease, such as tumor models. In addition, the protein of SEQ ID NO: 308 is structurally related to other proteins that show homology and / or structural similarity to human p27 (Rasmussen, UB et al., 199).
3, Cancer Research 53: 4096-4101, whose publication is incorporated herein by reference). Thus, brain, fetal brain, kidney, fetal kidney, or colon proteins can be used to control the proliferation of EPO-dependent cells, or the growth and development of erythroid and other hematopoietic lineages. The protein of SEQ ID NO: 308 or a fragment thereof, or a polynucleotide encoding the protein of SEQ ID NO: 308 or a fragment thereof, comprises anemia of chronic diseases and chronic renal failure, erythrocytosis, cancer, AIDS, drugs and phlebotomy-induced anemia, hemochromatosis, It can be used to treat or ameliorate erythropoiesis by EPO treatment, and other medical conditions associated with altered activity or levels of the protein of SEQ ID NO: 308. In another embodiment, the invention relates to methods of identifying agonists and antagonists / inhibitors using the protein of SEQ ID NO: 308 or fragments thereof, and treating the diseases with the identified compounds. In a further aspect, the invention relates to a diagnostic assay for detecting diseases associated with inappropriate protein levels or activity of SEQ ID NO: 308. Yet another embodiment of the invention is a protein of SEQ ID NO: 308, a fragment thereof, or a protein of SEQ ID NO: 308 for monitoring the value of iron therapy in patients who have EPO treatment, blood loss, or both. Alternatively, it relates to the use of the DNA encoding the fragment. DNA encoding the protein of SEQ ID NO: 308 or a fragment thereof is useful in a diagnostic assay for conditions / diseases associated with aberrant expression of the protein of SEQ ID NO: 308. Diagnostic assays are useful for identifying the absence, presence, and overexpression of the proteins of the invention, as well as monitoring regulatory levels of the proteins of the invention during therapeutic intervention. In addition, the DNA is introduced into an effective eukaryotic expression vector, gene therapy for the above-mentioned diseases including tumors, and / or pathologically or genetically-induced defects in any of the above-mentioned proteins including the proteins of the present invention. The correction can also be targeted directly to a specific tissue, organ, or cell population for use. In addition, DNA may be used to design antisense sequences and ribozymes that can be administered to alter gene expression in tumors and pathogen-infected cells, or to influence cytokine and growth factor expression. In vivo delivery of genetic constructs into a subject affects expression of the proteins of the invention or expression and / or expression of other proteins such as cytokines, growth factors, their receptors and / or tumor antigens.
Alternatively, it can be developed to the extent that it targets specific cell types such as tumors that modulate activity. In addition, detection of unknown upstream sequences (eg promoters and regulators) by conventional methods, and interferons and other cytokines,
It is also useful for studies on the regulation of gene expression by growth and transcription factors in normal and diseased cells. The hybridization probe is
Detection of DNA encoding the protein (or related molecule) of the invention in a biological sample,
And is useful for mapping naturally occurring genomic sequences to specific chromosomes / regions. DNA produces and / or treats in vivo animal models of disease, including infections, inflammations, tumors, autoimmune diseases, susceptibility or resistance to anemia and iron overload, and tumor therapy based on vaccines, knockouts and transgene technology. Can be used to

Antibodies to the protein of SEQ ID NO: 308 are useful in diagnosing diseases and disorders associated with its expression, and quantifying the proteins of the invention (eg, in assays that monitor patients during therapeutic intervention). Antibodies specific for proteins are
It may include, but is not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments produced by a Fab expression library. Neutralizing antibodies are particularly preferred for diagnostics and therapeutics. Diagnostic assays for the protein of SEQ ID NO: 308 include methods of detecting the protein of the invention in human body fluids or extracts of cells or tissues using antibodies and labels. The protein of SEQ ID NO: 308 and its catalytic or immunogenic fragments, or oligopeptides thereof, can be used to screen therapeutic compounds in any drug screening technique, including high throughput. Methods that can be used to quantify the expression of the nucleotides or proteins of the invention include polymerase chain reaction (PCR), RT-PCR, RNAse protection, Northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay. (RIA), fluorescence activated cell sorting (FA
CS), immunoprecipitation, and chromatography, but are not limited thereto. Accordingly, the invention provides a protein of SEQ ID NO: 308, at least 5, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, for treating or ameliorating a disease. 1
Including the use of fragments containing 50 or 200 consecutive amino acids, or fragments with favorable biological activity. For example, the disease is cancer including breast cancer, viral infection, bacterial infection, inflammation, autoimmune injury, rheumatoid arthritis, Graves' disease, systemic lupus erythematosus, autoimmune hepatitis, Wegener's granulomatosis, sarcoidosis, polyarthritis, celestial Pemphigus, pemphigoid, erythema multiforme, Sjogren's syndrome, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, scleritis, type I diabetes, insulin-dependent diabetes mellitus, lupus nephritis, and allergies Autoimmune disorders such as idiopathic encephalomyelitis; leukemia, lymphoma (Hodgkin and non-Hodgkin), sarcoma, melanoma, adenoma, solid tissue carcinoma, hypoxic tumor, squamous cell carcinoma of the mouth, pharynx, larynx, and lungs, neck And urogenital cancer, such as bladder cancer, hematopoietic cancer, head and neck cancer, and nervous system cancer,
Proliferative disorders including various forms of cancer such as papilloma, atherosclerosis, benign lesions such as angiogenesis, viral infections, especially HCV and HIV infections, and other pathogenic infections (eg Leishmania) Good.

In such an embodiment, the protein of SEQ ID NO: 308, or a fragment thereof, is administered to an individual who wishes to increase or decrease any of the activities of the protein of SEQ ID NO: 308. The protein of SEQ ID NO: 308 or a fragment thereof may be administered directly to the individual, or the nucleic acid encoding the protein of SEQ ID NO: 308 or a fragment thereof may be administered to the individual. Alternatively, a formulation that increases the protein activity of SEQ ID NO: 308 may be administered to the individual. Such a formulation is described in SEQ ID NO: 308.
Cells or a preparation containing the protein of SEQ ID NO: 308 or the protein of SEQ ID NO: 308 can be identified by contacting with a test agent and assaying whether the test agent increases the activity of the protein. For example, the test agent can be a chemical compound or polypeptide or peptide. Alternatively, the protein activity of SEQ ID NO: 308 can be reduced by administering to the individual a formulation that interferes with such activity. A formulation that interferes with the activity of the protein of SEQ ID NO: 308 is to contact the protein of SEQ ID NO: 308 or a cell or preparation containing the protein of SEQ ID NO: 308 with a test drug and test whether the test drug reduces protein activity. Can be identified by For example, the formulation can be a compound, polypeptide or peptide, antibody, or nucleic acid such as an antisense nucleic acid or a triple helix forming nucleic acid. In one embodiment, the invention provides for selective identification of the origin organs of a sample, such as brain, kidney, liver or cancer prostate, based on the protein level of SEQ ID NO: 308 in the sample, or of said sample. The present invention relates to a method and a composition using the protein of the present invention or a part thereof as a marker protein for distinguishing two or more origin vessels from each other. For example, the protein of SEQ ID NO: 308 or fragments thereof can be used to raise antibodies using techniques known to those of skill in the art, including those described herein. Such antibodies can then be used to identify tissues of unknown origin, such as tumor tissues that have metastasized and differentiated into, for example, forensic samples, different body sites, etc., or immunochemistry can be used to identify different tissue types in tissue sections. Can be used to distinguish. In such methods, the sample is contacted with a detectably labeled antibody under conditions that promote antibody binding. The level of antibody binding to the test sample is measured and compared to the level of binding to control cells derived from brain, kidney, pancreas or cancer prostate, or tissue other than brain, kidney, pancreas or cancer prostate, Determine from brain, kidney, pancreas or cancer prostate. Alternatively, the protein level of SEQ ID NO: 308 in the test sample may be measured by determining the level of RNA encoding the protein of SEQ ID NO: 308 in the test sample. RNA levels can be measured using methods such as nucleic acid arrays, or in situ hybridization, Northern blot, dot blot, or other methods known to those of skill in the art. If desired, an amplification reaction such as a PCR reaction may be performed on the nucleic acid sample prior to analysis. The RNA level in the test sample is compared to that of control cells derived from brain, kidney, pancreas or cancer prostate or tissue other than brain, kidney, pancreas or cancer prostate to determine whether the test sample is brain derived. judge.

In another embodiment, antibodies to the protein of the invention or portions thereof are used for the detection, enrichment, or purification of cells expressing the protein of SEQ ID NO: 308, including techniques known to those of skill in the art. Can be used. For example, an antibody to the protein of SEQ ID NO: 308 or a fragment thereof can be immobilized on a solid support such as a chromatography matrix. A preparation comprising cells expressing the protein of SEQ ID NO: 308 is contacted with the antibody under conditions that promote binding to the antibody. After washing the support, the cells are released from the support by contacting the support with a drug that dissociates the cells from the antibody. In another embodiment of the invention, the protein of SEQ ID NO: 308 or a fragment thereof has a protein level of SEQ ID NO: 308 in a diseased individual that can be used to diagnose disorders associated with altered expression of the protein of SEQ ID NO: 308. Is measured using a method such as those described herein. In such methods, the protein level of SEQ ID NO: 308 in a diseased individual is compared to the level in a healthy individual to determine if the individual has a protein level of SEQ ID NO: 308 associated with the disease. <SEQ ID NOS: 289 and 307 proteins (internal designation numbers 175-1-3-0-E5-CS.cor and 187-39-0-0-k12-CS)> SEQ ID NOS: 289 protein is SEQ ID NO: 48 It is encoded by some cDNA.
Accordingly, all features and uses of the polypeptide of SEQ ID NO: 289 described throughout this application also relate to the polypeptide encoded by the human cDNA which is clone 175-1-3-0-E5-CS. It will be understood. Furthermore, it is understood that all features and uses of the nucleic acid of SEQ ID NO: 48 described throughout this application relate to the nucleic acid encoded by the human cDNA which is clone 175-1-3-0-E5-CS. Will be done. The protein of the present invention consists of 130 amino acids. According to amino acid alignment and hydrophobicity plot, the protein showed that the predicted signal peptide sequence for 8 to 20 residues and the predicted membrane for 4 residues of 2 to 24, 42 to 61, 70 to 90 and 99 to 119. It has a transmembrane domain. Thus, some embodiments of the present invention relate to polypeptides that include a signal peptide and / or one or more transmembrane domains.

The protein of SEQ ID NO: 289, which is encoded by the cDNA of SEQ ID NO: 48, is a human secretory protein in SEQ ID NO: 4199 of EP 1033401-A2 (the publication of which is incorporated herein by reference). Is homologous. SEQ ID NO: another protein
SEQ ID NO: 307, encoded by the 66 cDNA, is a polymorphic variant of the protein of SEQ ID NO: 289 and has all the functions and uses described herein. The present invention provides a novel protein identified from among cDNAs of a library prepared from salivary glands, and nucleic acid and amino acid sequences disclosed in the present specification, for disease research,
It relates to applications in diagnosis, prevention and treatment. Analysis of the tissue distribution predicted by BLAST in the database shows that the mRNA encoding this protein was detected mainly in brain and fetal brain, and to a lesser extent in kidney, fetal kidney and colon. Interferons (IFNs) are part of a group of intercellular messenger proteins known as cytokines. α-IFN is the product of a multigene family of at least 16 members, while b-IFN is the product of a single gene. Furthermore, α- and β-IFN are also known as type I IFN. Type I IFN is produced in various cell types. Type I IFN biosynthesis is stimulated by viruses and other pathogens, and diverse cytokines and growth factors. Γ-IFN, also known as type II IFN, is produced in T cells and natural killer cells. The antigen used to sensitize the organism stimulates type II IFN biosynthesis.
α- and γ-IFN are both immune modulators and anti-inflammatory agents, activating macrophages, T cells and innate killer cells. IFN is part of the body's natural defenses against viruses and tumors. IFNs exert their defense functions by affecting the functioning of the immune system and by direct action on pathogens and tumor cells. IFNs mediate some of these multiple effects by inducing the synthesis of many cellular proteins. Some interferon inducible (IFI) genes are induced in much the same way by by α-, β- and γ-IFN. Other IFI genes are preferentially induced by type I or type II IFN. The various proteins produced by the IFI gene are antitumor,
It has antiviral and immunomodulatory functions. Tumor antigen expression in cancer cells is
Increased by IFN, making cancer cells more susceptible to immune rejection. IFI proteins synthesized in response to viral infection are known to suppress viral functions such as cell penetration, decoating, RNA and protein synthesis, assembly and release (Hardman JG et al. 25 (1996) The Pharmacological Basis. of Th
erapeutics, McGraw-Hill, New York NY pp 1211-1214, the entire disclosure of which is incorporated herein by reference). Type II IFN stimulates expression of the major histocompatibility complex (MHC) protein and is therefore used to enhance the immune response.

The protein of SEQ ID NO: 289 is a low molecular weight hydrophobic protein, isoforms 6-16 of human interferon inducible (IFI) protein (97% identity).
, HIFI (44%) and p27 (33%), and the chimpanzee homologue of 6-16, 130-51- (97%), show chemical and structural homology. Therefore,
The protein of SEQ ID NO: 289 and its encoding nucleic acid are polymorphic variants of the gene encoding 6-16 or 6-16. The protein of SEQ ID NO: 289, a fragment thereof, or the nucleic acid encoding the protein of SEQ ID NO: 289 or a fragment thereof can be used in the diagnosis, study, prevention and treatment of diseases as described below. The IFI gene, known as 6-16, encodes an mRNA that is highly induced by type I IFN in various human cells (Kelly JM et al. (1986) EMBO J 5:16.
01-1606, the entire text of which is incorporated herein by reference). After induction, 6-16 mRNA accounts for 0.1% of total cell mRNA. 6-16 mRNA is only present at very low levels in the absence of type I IFN and is slightly induced by type II IFN. 6-16
mRNA encodes a 130 amino acid hydrophobic protein. The first 20-23 amino acids contain a putative signal peptide. The 6-16 protein has at least two predicted transmembrane regions ending in a negatively charged C-terminus. The p27 gene encodes a protein that exhibits 41% amino acid sequence identity with the 6-16 protein. The p27 gene is expressed in several breast tumor cell lines and gastric cancer cell lines. In other breast tumor cell lines, HeLa cervical cancer cell lines, and fetal lung fibroblasts, p27 expression occurs only by α-IFN induction. In one breast tumor cell line, p27 is separately induced by estradiol and IFN (Rasmussen UB et al. (1993) Cancer Res 53: 4096-4101, the publication of which is incorporated herein by reference in its entirety. ing). Expression of p27 was analyzed in 21 primary invasive breast cancers, 1 breast cancer bone metastasis, and 3 breast fibroadenoma. High levels of p27 were detected in about half the primary carcinoma and bone metastases but not in fibroadenoma. These observations suggest that some breast tumors produce high levels of type I IFN or have an increased susceptibility to type I IFN as compared to other breast tumors ( Rasmussen UB et al., Supra). Furthermore, the p27 gene is expressed at significant levels in normal tissues including colon, stomach and lung, but not in placenta, kidney, liver or skin. (Rasmussen UB et al., Supra).

Small products of the hydrophobic IFI gene may contribute to viral resistance. The hepatitis C virus (HCV) -inducible gene, 130-51, was isolated from a cDNA library prepared from chimpanzee liver during the acute phase of infection. The protein product of this gene is
97% identity to human 6-16 protein (Kato T et al. (1992) Virology 190: 85
6-860, the full text of which is incorporated herein by reference). The authors of the aforementioned article suggest that HCV infection actively induces IFN expression, which in turn induces expression of the IFI gene, including 130-51. IFI proteins synthesized in response to viral infection are known to suppress viral functions such as cell penetration, decoating, RNA and protein synthesis, assembly and release. The 130-51 protein may suppress one or more of these functions in HCV. The IFI protein can suppress multiple functions of a particular virus. Furthermore, the major inhibitory effects on IFI proteins vary by virus family (Hardman JG, supra, p1211, the publication of which is incorporated herein by reference). The HIFI protein (International Patent Application Publication WO 9812223-A2, the publication of which is incorporated by reference in its entirety) is a human sequence identified from the cDNA of a library prepared from human neonatal kidney. Is. LIFESEQTM database (Inc
yte Pharmaceuticas, Palo Alto, CA) Northern blot analysis using HIFI
It shows that mRNA was detected only in the neonatal kidney. HIFI protein is 104
Consists of amino acids of p27, 6-16 and 130-51, 55%, 45%, and 4 respectively.
It shows 6% amino acid sequence identity. The hydrophobic IFI proteins of the invention may be the basis for clinical diagnosis of diseases associated with their induction. These proteins can be tumors, viral infections, inflammation, or
It may be useful in the diagnosis and treatment of pathologies associated with impaired immunity. Furthermore, these proteins can be used to study the regulation of gene expression by IFN and other cytokines, as well as hormones and growth factors, normal and diseased cells. Based on the chemical and structural homology between the protein of SEQ ID NO: 289 and the hydrophobic IFI small protein of human and chimpanzee, the protein of SEQ ID NO: 289 is produced by interferon in infection, inflammation, autoimmune disease, etc. It seems to be synthesized when it is done. Interferons are produced in response to diverse cytokines and growth factors in viral infections, inflammation, autoimmune diseases, and cancer. Therefore, the protein of SEQ ID NO: 289 or a fragment thereof is
It can be used to diagnose and treat diseases including, but not limited to, rheumatoid arthritis, Graves' disease, systemic lupus erythematosus, autoimmune hepatitis, Wegener's granulomatosis, sarcoidosis, polyarthritis, pemphigus. , Pemphigoid, erythema multiforme, Sjogren's syndrome, inflammatory bowel disease, multiple sclerosis, myasthenia gravis keratitis, scleritis, type I diabetes, insulin-dependent diabetes mellitus, lupus nephritis, and allergic Autoimmune disorders such as encephalomyelitis; leukemias, lymphomas (Hodgkin and non-Hodgkins), sarcomas, melanomas, adenomas, solid tissue carcinomas, hypoxic tumors, squamous cell carcinomas of the mouth, pharynx, larynx, and lungs, neck and neck Urogenital cancer such as bladder cancer, hematopoietic cancer, head and neck cancer, and nervous system cancer, papilloma, atherosclerosis, benign injury such as angiogenesis, Proliferative disorders including various forms of cancer such as; viral infections,
Especially HCV and HIV infections, as well as other pathogenic infections (eg Leishmania).

Further, the protein of SEQ ID NO: 289 or fragments thereof can be used to treat diseases associated with inflammation or immune dysfunction (eg rheumatism and osteoarthritis and AIDS). Another embodiment of this invention is directed to the use of the protein of SEQ ID NO: 289 or a fragment thereof to treat and / or prevent the disease effects of bacterial infection in a pregnant mammal, such as spontaneous abortion and maternal death. . In a preferred embodiment, the proteins of the invention can be used to counteract the effects of bacterial endotoxin lipopolysaccharide (LPS). Methods for using such compositions are described in Dziegiel
ewska and Andersen, Biol. Neonate, 74: 372-5 (1998),
The entire publication is incorporated herein by reference. Furthermore, the protein of SEQ ID NO: 289 or a fragment thereof is useful as a reagent for analyzing the regulation of gene expression by interferons and other cytokines in both normal and diseased cells. The protein of SEQ ID NO: 289 or fragments thereof can be used to identify specific molecules such as agonists, antagonists or inhibitors that bind to it. Another embodiment of the invention is SEQ ID NO: 289 to identify and / or quantify the interferon family of cytokines and other cytokines such as IL-10 and tumor antigens that may interact with the proteins of the invention. Of the protein or a fragment thereof. In addition, the protein of SEQ ID NO: 289 or a fragment thereof may be contained in a pharmaceutical preparation for the treatment of cancer or the prevention / treatment of other diseases associated with altered expression of the proteins of the invention. In another embodiment of the invention, the protein of SEQ ID NO: 289 or a fragment thereof prevents the binding of endogenous cytokines to their natural receptors, which results in regulatory signals generated by cell proliferation or ligand receptor binding events. Blocking cytokines and Il-2, TNFalpha, CTL
It can be used to suppress and / or modulate the effects of A4, CD28, and other related molecules. The protein of SEQ ID NO: 289 or a fragment thereof is an in vivo model of disease, such as an autoimmune, inflammation and tumor model, by injecting the protein into the affected tissue by intraperitoneal, intravenous, subcutaneous or direct methods. It is useful to correct the defects in.

DNA encoding the protein of SEQ ID NO: 289, or fragments thereof, is useful in diagnostic assays for diseases / disorders associated with expression of the proteins of the invention. Diagnostic assays are useful for identifying the absence, presence, and overexpression of the proteins of the invention, as well as for monitoring regulatory levels of the proteins of the invention during therapeutic intervention.
Further, the DNA is introduced into an effective eukaryotic expression vector for gene therapy of the above-mentioned diseases including tumors and / or correction of pathological or genetically induced defects in any of the above-mentioned proteins including the protein of the present invention. , Can also be targeted directly to a specific tissue, organ, or cell population for use. In addition, DNA may be used to design antisense sequences and ribozymes that can be administered to alter gene expression in tumors and pathogen-infected cells, or to influence cytokine and growth factor expression. . In vivo delivery of genetic constructs into a subject affects the expression of the proteins of the invention or modulates the expression and / or activity of other proteins such as cytokines, growth factors, their receptors and / or tumor antigens. It can be developed to the extent that it is targeting specific cell types, such as tumors, that are stimulating. In addition, unknown upstream sequences (eg promoters and regulators) can be routinely detected to study interferon and other cytokines as well as control of gene expression by growth and transcription factors in normal and diseased cells. It is useful. Hybridization probes are useful for detecting DNA encoding a protein (or related molecule) of the invention in a biological sample and for mapping naturally occurring genomic sequences to specific chromosomes / chromosomal regions. DNA
Can be used to produce and / or treat in vivo animal models of disease, including susceptibility or resistance to infections, inflammation, tumors and autoimmune diseases, and tumor therapy based on vaccines, knockouts and transgene technology. . Antibodies to the protein of SEQ ID NO: 289, or fragments thereof, are useful for diagnosing diseases and disorders associated with its expression, and for quantifying the proteins of the invention (eg, in assays that monitor patients during therapeutic intervention). Antibodies specific for the protein may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments produced by the Fab expression library. Neutralizing antibodies are particularly preferred for diagnostics and therapeutics. Diagnostic assays for the proteins of the invention include methods of detecting the proteins of the invention in human body fluids or cell or tissue extracts using antibodies and labels.

The proteins of the invention and their catalytic or immunogenic fragments, or oligopeptides thereof, can be used to screen therapeutic compounds in any drug screening technique, including high throughput. Methods that can be used to quantify the expression of the nucleotides or proteins of the invention include polymerase chain reaction (PCR), RT-PCR, RNAse protection, Northern and Western blots, enzyme-linked immunosorbent assay (ELISA), Includes, but is not limited to, radioimmunoassay (RIA), fluorescence activated cell sorting (FACS), immunoprecipitation, and chromatography. Accordingly, the invention provides a protein of SEQ ID NO: 289, at least 5,8,10,12,15.
, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150 or 200 contiguous amino acid fragments, or in an individual for treating or ameliorating a disease,
Including uses of fragments having the desired biological activity. For example, the disease is cancer including breast cancer, viral infection, bacterial infection, inflammation, autoimmune injury, rheumatoid arthritis, Graves' disease, systemic lupus erythematosus, autoimmune hepatitis, Wegener's granulomatosis, sarcoidosis, polyarthritis, celestial Pemphigus, pemphigoid, erythema multiforme, Sjogren's syndrome, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, scleritis, type I diabetes, insulin-dependent diabetes mellitus, lupus nephritis, and allergies Autoimmune disorders such as idiopathic encephalomyelitis; leukemia, lymphoma (Hodgkin and non-Hodgkin), sarcoma,
Melanoma, adenomas, solid tissue carcinomas, hypoxic tumors, squamous cell carcinomas of the mouth, pharynx, larynx, and lungs, genitourinary cancers such as cervical and bladder cancers, hematopoietic cancers, head and neck cancers, and nervous system Proliferative disorders including various forms of cancer such as cancer, papilloma, atherosclerosis, benign lesions such as angiogenesis; viral infections, especially HCV and HIV infections, and other pathogenic infections (eg Leishmania) Including. In such embodiments, the protein of SEQ ID NO: 289, or a fragment thereof, is administered to an individual who desires to increase or decrease any of the activities of the protein of SEQ ID NO: 289. The protein of SEQ ID NO: 289 or fragment thereof may be administered directly to the individual, or a nucleic acid encoding the protein of SEQ ID NO: 289 or fragment thereof may be administered to the individual. Alternatively, a formulation that increases the protein activity of SEQ ID NO: 289 may be administered to the individual. Such formulations can be identified by contacting the protein of SEQ ID NO: 289 or cells or preparations containing the protein of SEQ ID NO: 289 with a test agent and assaying whether the test agent increases the activity of the protein. For example, the test agent can be a chemical compound or polypeptide or peptide.

Alternatively, the protein activity of SEQ ID NO: 289 may be reduced by administering to the individual a formulation that interferes with such activity. Formulations that interfere with the activity of the protein of SEQ ID NO: 289 contact cells or preparations containing the protein of SEQ ID NO: 289 or the protein of SEQ ID NO: 289 with a test agent and determine whether the test agent reduces protein activity. It can be identified by For example, the formulation as used herein may include a compound, polypeptide or peptide, antibody, or nucleic acid such as antisense nucleic acid or triple-helix forming nucleic acid. In one embodiment, the present invention provides for selectively identifying a source organ of a sample, such as brain, fetal brain, kidney, fetal kidney or colon, based on the protein level of SEQ ID NO: 289 in the sample. Or a method of using the protein of the present invention or a part thereof as a marker protein for distinguishing two or more origin vessels of the sample from each other. For example, the protein of SEQ ID NO: 289 or fragments thereof can be used to raise antibodies using techniques known to those of skill in the art, including those described herein. Such antibodies may then identify tissue of unknown origin, such as tumor tissue that has metastasized and differentiated into forensic samples, different body sites, etc., or may use immunochemistry to identify different tissue types in the tissue section. Can be used to distinguish. In such methods, the sample is contacted with a detectably labeled antibody under conditions that promote antibody binding. The level of antibody bound to the test sample is measured to determine the brain, fetal brain, kidney, fetal kidney or colon,
Alternatively, determine whether the test sample is derived from brain, fetal brain, kidney, fetal kidney or colon by comparing the level of binding to control cells derived from tissues other than brain, fetal brain, kidney, fetal kidney or colon . Alternatively, the protein level of SEQ ID NO: 289 in the test sample may be measured by determining the RNA level encoding the protein of SEQ ID NO: 289 in the test sample. RNA levels can be measured using methods such as nucleic acid arrays or in situ hybridization, Northern blots, dot blots, or other methods known to those of skill in the art. If desired, an amplification reaction such as a PCR reaction may be performed on the nucleic acid sample prior to analysis. RNA levels in the test sample are compared to those of control cells derived from brain, fetal brain, kidney, fetal kidney or colon or tissues other than brain, fetal brain, kidney, fetal kidney or colon. , Fetal brain, kidney, fetal kidney or colon.

In another embodiment, antibodies to the proteins of the invention or portions thereof are used to detect, enrich, or purify cells expressing the protein of SEQ ID NO: 289, including techniques known to those of skill in the art. Can be used. For example, an antibody to the protein of SEQ ID NO: 289 or a fragment thereof can be immobilized on a solid support such as a chromatography matrix. A preparation comprising cells expressing the protein of SEQ ID NO: 289 is contacted with the antibody under conditions that promote binding to the antibody. The cells are released from the support by washing the support and then contacting the support with a drug that causes the cells to dissociate from the antibody. In another embodiment of the invention, the protein of SEQ ID NO: 289 or a fragment thereof can be used to diagnose disorders associated with altered expression of the protein of SEQ ID NO: 289. In such methods, the protein level of SEQ ID NO: 289 in diseased individuals is measured using methods such as those described herein. The protein level of SEQ ID NO: 289 in a diseased individual is compared to the level in a healthy individual to determine if the individual has a protein level of SEQ ID NO: 289 associated with the disease. <Protein of SEQ ID NO: 268 (Internal Designation Number 116-111-4-0-B3-CS) The protein of SEQ ID NO: 268 is encoded by the cDNA of SEQ ID NO: 27.
Accordingly, all features and uses of the polypeptide of SEQ ID NO: 268 described throughout this application also apply to the polypeptide encoded by the human cDNA, which is clone 116-111-4-0-B3-CS. It will be understood that this is the case. Furthermore, all features and uses of the nucleic acid of SEQ ID NO: 27 described elsewhere in this application also relate to the nucleic acid encoded by the human cDNA which is clone 116-111-4-0-B3-CS. Will be understood. The proteins of the invention have been found to be expressed in the testes and lungs. The protein of SEQ ID NO: 268, encoded by the 27 extended cDNA of SEQ ID NO: 268, is a member of the CT antigen family and is overexpressed in Ewing sarcoma.
-1 is a splicing variant of -1 (Liu, XF, LJ Helman et al., (2000) Canc.
er Res 60 (17): 4752-5, the disclosure of which is incorporated herein by reference in its entirety). In addition, the protein of SEQ ID NO: 268 has a CT at the COOH terminus.
Strong homology to PAGE4, another member of the antigen family (Brinkmann,
U., G. Vasmatzis et al., (1994) Cancer Res 59 (7): 1445-8, the disclosure of which is incorporated herein by reference in its entirety).

The cDNA of SEQ ID NO: 27 consists of 5 exons. Exon 1 is nucleotides 1-2
45, exon 2 nucleotides 246-370, exon 3 nucleotides 371-512, exon 4 at nucleotides 513-639, and exon 5 at nucleotides 640-762.
Exons 2 to 5 of the cDNA of SEQ ID NO: 27 are partially shared with XAGE-1. However, there is a frameshift in the alignment of the two molecules because the start codon of SEQ ID NO: 27 is located within intron 1 of XAGE-1. Exon 1 of SEQ ID NO: 27 is at nucleotides 110-234 of XAGE-1, exon 2 of SEQ ID NO: 27 is at nucleotides 235-376 of XAGE-1, and exon 3 of SEQ ID NO: 27 is nucleotides 377-503 of XAGE-1.
And exon 4 of SEQ ID NO: 27 is at nucleotides 504 to 526 of XAGE-1. XAGE-1 is overexpressed in sarcoma and alveolar rhabdomyosarcoma and highly expressed in normal testis (Liu, XF, LJ Helman et al. (2000). Cancer Res.
60 (17): 4752-5, the entire disclosure of which is incorporated herein by reference. Furthermore, XAGE-1 shows homology to PAGE-4 at the COOH terminus (Brinkmann, U.
, G. Vasmatzis, et al. (1999) Cancer Res 59 (7): 1445-8, the entire disclosure of which is incorporated herein by reference. CT antigen is expressed by cancers that occur in non-essential normal tissues such as the prostate, breast, and ovary (G. Vasmatzis et al., Proc. Natl. Acad. Sci. USA, 95: 300-304.
, 1998, the disclosure of which is incorporated herein by reference in its entirety), and which has a distinct expression pattern in normal tissues and is a different class of differentiation antigens. This class of antigens is presented on the surface of tumor cells, recognized by cytolytic T cells, and ultimately lysed. The extent to which these antigens have been studied is through characterization studies of cytolytic T cells in vitro, i.e. identification of a subset of specific cytolytic T cells (hereinafter "CTL"). This subset is
Cells that proliferate upon recognition of the presented tumor rejection antigen and lyse the antigen-presenting cells are lysed. Characterization studies have identified CTL clones that specifically lyse cells expressing the antigen. An example of this study is Levy et al., Adv. Cancer Res. 24: 1-59 (1977).
Boon et al., J. Exp. Med. 152: 1184-1193 (1980); Brunner et al., J. Immunol. 12
4: 1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 124: 1627-1634 (1980.
); Maryanski et al., Eur. J. Immunol. 12: 406-412 (1982); Palladino et al., Canc.
Res. 47: 5074-5079 (1987), the disclosures of each is incorporated by reference in its entirety.

Thoroughly studied CT antigens include MAGE, BAGE, GAGE and LAGE
, XAGE, and other antigens, most of which are located on the X chromosome. Brinkma
nn et al. reported the identification of three new members of the GAGE / PAGE family called XAGE. XAGE-1 and XAGE-2 are expressed in Ewing sarcoma, rhabdomyosarcoma, breast cancer, and germ cell tumors. The protein of SEQ ID NO: 268 appears to be a splicing variant of XAGE-1, a CT antigen overexpressed in Ewing sarcoma. Accordingly, the invention provides a protein of SEQ ID NO: 268, at least 5, 8, 10, 12, 15
, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150 or 200 contiguous amino acid fragments or associated with under-expression of the protein of SEQ ID NO: 268, as listed above. The use of fragments having the desired biological activity to treat or ameliorate the pathologies In such embodiments, the protein of SEQ ID NO: 268, or a fragment thereof, is administered to an individual who wishes to increase or decrease any of the activities of the protein of SEQ ID NO: 268. The protein of SEQ ID NO: 268 or fragment thereof may be administered directly to an individual, or the nucleic acid encoding the protein of SEQ ID NO: 268 or fragment thereof may be administered to an individual. Alternatively, a formulation that increases the protein activity of SEQ ID NO: 268 may be administered to the individual. Such formulations can be identified by contacting the protein of SEQ ID NO: 268 or cells or preparations containing the protein of SEQ ID NO: 268 with a test agent and analyzing whether the test agent increases the activity of the protein. For example, the test agent can be a chemical compound or polypeptide or peptide. Alternatively, the protein activity of SEQ ID NO: 268 can be reduced by administering to the individual a formulation that interferes with such activity. Formulations that interfere with the activity of the protein of SEQ ID NO: 268 contact a cell or preparation containing the protein of SEQ ID NO: 268 or the protein of SEQ ID NO: 228 with a test agent and determine whether the test agent reduces protein activity. It can be identified by For example, the formulation here is a compound,
Mention may be made of polypeptides or peptides, antibodies, or nucleic acids such as antisense nucleic acids or triple-helix forming nucleic acids. In one embodiment, the invention provides for the selective identification of tissues, preferably the testes and lungs, or the two source organs of said sample, based on the protein level of SEQ ID NO: 268 in the sample. The present invention relates to a method and a composition using the protein of the present invention or a part thereof as a marker protein for identifying them when they are exchanged. For example, the protein of SEQ ID NO: 268 or fragments thereof can be used to raise antibodies using techniques known to those of skill in the art, including those described herein. Such tissue-specific antibodies can then be used to identify tissue of unknown origin, such as forensic samples, tumor tissue that has metastasized and differentiated into different body sites, etc., or different tissue types in tissue sections using immunochemistry. Can be used to distinguish In such methods, a detectably labeled antibody is contacted with a tissue sample under conditions that promote antibody binding. The level of antibody binding to the test sample is measured and compared to the level of binding to test cells or lungs or control cells from tissues other than testis or lungs to determine whether the test sample is from testes or lungs. Alternatively, the protein level of SEQ ID NO: 268 in the test sample may be measured by determining the RNA level encoding the protein of SEQ ID NO: 268 in the test sample. RNA levels can be measured by nucleic acid array or in situ hybridization, Northern blot,
It can be measured using a method such as dot blot or other methods known to those skilled in the art. If desired, an amplification reaction such as a PCR reaction may be performed on the nucleic acid sample prior to analysis. The RNA level in the test sample is compared to that of control cells from the testis or lung or testis or tissue other than lung to determine whether the test sample is from the testis or lung.

In other embodiments, antibodies to the proteins of the invention, or portions thereof, can be used to treat Ewing sarcoma cells, rhabdomyosarcoma cells, breast cancer cells and sex cell carcinoma cells using techniques known to those of skill in the art. Including, it can be used for the detection, enrichment, or purification of cells expressing the protein of SEQ ID NO: 268. For example, an antibody against the protein of SEQ ID NO: 268 or fragments thereof can be immobilized on a solid support such as a chromatography matrix. A preparation comprising cells expressing the protein of SEQ ID NO: 268 is contacted with the antibody under conditions that promote binding to the antibody. After washing the support, the cells are released from the support by contacting the support with a formulation that dissociates the cells from the antibody. In another embodiment of the invention, the protein of SEQ ID NO: 268 or fragments thereof can be used to diagnose disorders associated with altered expression of the protein of SEQ ID NO: 268. In some embodiments, the protein of SEQ ID NO: 268 or fragments thereof can be used to diagnose Ewing sarcoma, rhabdomyosarcoma, breast cancer and germ cell tumors. In such methods, the protein level of SEQ ID NO: 268 in a diseased individual is measured using a method such as those described herein. The protein level of SEQ ID NO: 268 in diseased individuals is compared to the level in healthy individuals. An increase or decrease in the protein level of SEQ ID NO: 268 compared to a healthy individual suggests that the sick individual suffers from a defect in intercellular communication or secretion. Other embodiments of the invention relate to compositions and methods that use the protein of SEQ ID NO: 268 or fragments thereof as potential targets for vaccine therapy of cancers including Ewing sarcoma, rhabdomyosarcoma, breast cancer or germ cell tumors. . In such an embodiment, an individual afflicted with cancer is administered an antibody against the protein of SEQ ID NO: 268 or fragments thereof in an amount sufficient to ameliorate or eliminate the cancer. <Protein of SEQ ID NO: 399 (Internal Designation No. 160-40-1-0-H4-CS)> The protein of SEQ ID NO: 399 is encoded by the cDNA of SEQ ID NO: 158.
Therefore, it is understood that all features and uses of the polypeptide of SEQ ID NO: 399 described throughout this application also relate to the polypeptide encoded by the human cDNA which is clone 160-40-1-0-H4-CS. Will. In addition, all features and uses of the nucleic acid of SEQ ID NO: 158 described throughout this application are also provided by clone 160-40-1-0-H4.
It will be appreciated that it relates to the nucleic acid encoded by the human cDNA which is -CS. The proteins of the invention have been found to be expressed in the testis and lung. This protein is present in excess in fetal brain. The protein of SEQ ID NO: 399 encoded by the cDNA of SEQ ID NO: 158 is a protein of the phosphatidic acid phosphatase type 2 (PAP2) superfamily (Stukey
J. and Carman GM, Protein Sci 1997; 6: 469-472, the disclosure of which is incorporated herein by reference). Three human PAP variants, PAP-alpha 2 (W79285) and its alternatively spliced PAP
-alpha 1 (W79284), PAP-beta (W79286) and PAP-gamma (W79287) have been identified. The protein of SEQ ID NO: 399 displays the pfam characteristic domain of the PAP2 superfamily at positions 19-175. Accordingly, one embodiment of the invention is a polypeptide comprising amino acid residues 19-175 of SEQ ID NO: 399.

Phosphatidic acid phosphatase (PAP) (also called phosphatidic acid phosphate hydrolase) has been identified as an important enzyme in the biosynthesis of glycerolipids. In particular, PAP catalyzes the conversion of phosphatidic acid (PA) to diacylglycerol (DAG). PA and DAG are lipids involved in cell signaling and biosynthesis of structural membrane lipids and are therefore important control points in the metabolism of eukaryotic phospholipids. DAG is a widely studied lipid second messenger and is essential for the activation of protein kinase C, whereas (Kent; Anal. Rev. Biochem.
64: 315-343; 1995; PA, which is also a lipid messenger, is involved in various signal transduction pathways such as NADPH oxidase activation and calcium mobilization (English; Cell Signal .; 8: 341- 347; 1996, the entire publication of which is incorporated herein by reference). Therefore, regulation of PAP activity depends on PA and DAG.
Affects the balance of divergent signaling processes that cells receive (Brindl
ey et al .; Chem. Phys. Lipids 80: 45-57; 1996, the entire disclosure of which is incorporated herein by reference). PAP exists in at least two isoforms, one (PAP1) is cytoplasmic and membrane-associated and the other (PAP2) is a putative integral membrane protein (Leung DW, Tompkins CK, White). T .; DNA Cell Biol. 17: 377-385 (
1998)). The protein of the present invention has 180 amino acids and four predicted transmembrane segments and is therefore presumed to be an integral membrane protein. The protein of SEQ ID NO: 399 is encoded by a cDNA that is homologous to multiple alternatively spliced forms of the PAP2 gene. For example, the protein of SEQ ID NO: 399 shows 29% homology to human phosphatidic acid phosphatase type 2C protein. SEQ ID NO: 399 also shows 40% homology to the human phosphatidic acid dephosphatase 2B protein. In addition, the protein of SEQ ID NO: 399 shows 33% homology to the human phosphatidic acid phosphatase alpha-22 type protein. PAP2
-alpha2 is one of two presumed isoforms consisting of PAP2-alpha1 and a single gene-derived alternative splicing variant. Northern analysis revealed that PAP2-alpha mRNA expression was suppressed in tumor tissue, indicating that PAP-2 may act as a tumor suppressor. P
The relationship between AP and tumor suppression was further substantiated by the finding that PAP activity was lower than that of the parental rat1 cell line in fibroblast cell lines transformed with the ras or fps oncogene (Brindley et al., Chem. . Phys. Lipids 80: 45-57; 199
The full text of the publication is incorporated into the present specification as a reference). As explained above, a decrease in PAP activity in transformed cells correlates with an accompanying increase in PA concentration. Furthermore, increased PAP activity and reduced levels of PA were found in contact-inhibited fibroblasts compared to proliferating and transformed fibroblasts (Brindl
ey et al., Chem. Phys. Lipids 80: 45-57; 1996, the entire disclosure of which is incorporated herein by reference). Thus, the protein of SEQ ID NO: 399 or fragments thereof can be used to reduce cell division, thereby providing a useful tool for treating cancer. Subsequent analysis of colon tumor tissue from four donors confirmed that PAP2-alpha expression was subordinate to the corresponding healthy colon tissue. Given these data and previous evidence that some transformed cell lines have reduced PAP activity, human PAP cDNA can be used for gene therapy of several tumors (Leung DW, Tompkins CK, White T. ； DNA
Cell Biol. 17: 377-385 (1998), the entire disclosure of which is incorporated herein by reference). Therefore, one embodiment of the present invention is the use of the protein of SEQ ID NO: 399 or a fragment thereof as a tumor suppressor. For example,
A nucleic acid expressing the protein of SEQ ID NO: 399 or a fragment thereof may be introduced into an individual suffering from cancer in order to ameliorate or eliminate the cancer. In fact, human phosphatidic acid phosphatase-encoding nucleic acids have been used in the control of levels of lipid cell mediators and in gene therapy such as cancer gene therapy (International Patent Application Publication No. WO 98/46730, the disclosure of which is fully incorporated herein). Are incorporated herein by reference).

In another embodiment of the invention, the protein of SEQ ID NO: 399 or a fragment thereof can be used to control the lipid mediator balance of cell activation and signaling. The protein of the present invention is human phosphatidic acid phosphatase 2A.
It shows 33% homology to the protein. PAP2A is a cell surface integral membrane glycoprotein that plays an active role in the hydrolysis and uptake of extracellular space lipids (Roberts RZ, Morris AJ; Biochim Biophys Acta 2000 Aug 24; 1487).
(1): 33-49, the full text of which is incorporated herein by reference. Accordingly, methods such as those described herein are used to modulate the level or activity of the protein of SEQ ID NO: 399 to affect the rate or extent of extracellular space lipid hydrolysis and uptake. be able to. In another embodiment of this invention, the protein of SEQ ID NO: 399 can be used to counter the inflammatory response. PA is a cytokine-induced inflammatory response (Bursten et al .; Circ. Sho
k 44: 14-29, 1994; Abraham et al .; J. Exp. Med. 181: 569-575, 1995; Rice et al .; P.
NAS 91: 3857-3861, 1994; Leung et al .; PNAS 92: 4813-4817, 1995, the disclosures of which are incorporated herein by reference in their entirety), and many involved in signal transduction. Of protein phosphatase in Escherichia coli (English et al., Chem. Phys. L
ipids 80: 117-132, 1996, the disclosures of which are allegedly incorporated herein by reference. Furthermore, the nucleic acid encoding the protein of SEQ ID NO: 399 or a fragment thereof, degrades excess intracellular PA,
It can be used to counteract the inflammatory response due to cytokine stimulation or to treat or ameliorate inflammatory diseases. The gene encoding the protein of SEQ ID NO: 399 or a fragment thereof can be used in gene therapy for the treatment of obesity associated with diabetes. PAP activity was reduced in the liver and heart of overweight obese and insulin-resistant JCR: LA obese rats compared to the lean control rat phenotype (Brindley et al. Chem. Phys. Lipid.
s 80: 45-57; 1996, the entire disclosure of which is incorporated herein by reference). Thus, the proteins of the present invention can provide important tools for the treatment of obesity associated with diabetes. Accordingly, the invention provides a protein of SEQ ID NO: 399, at least 5 of that protein.
, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150 or 200 contiguous amino acid fragments, or in an individual, a disease as described above Includes the use of fragments having the desired biological activity to treat or ameliorate. In such embodiments, the protein of SEQ ID NO: 399 or a fragment thereof is administered to an individual who desires to increase or decrease any of the activities of the protein of SEQ ID NO: 399. Here, the activity of the protein includes the following: glycerolipid biosynthesis, conversion of phosphatidic acid to diacylglycerol, signal transduction, membrane lipid biosynthesis, activation of protein kinase C, NADPH oxidase Activation, calcium mobilization, cell division, diacylglycerol, monoacylglycerol, ceramide or sphingosine production, inflammatory response or dephosphorylation of substrates such as lysophosphatidic acid, ceramide 1-phosphate, or sphingosine 1-phosphate Or the treatment or amelioration of obesity associated with diabetes. The protein of SEQ ID NO: 399 or a fragment thereof may be administered directly to an individual, or the nucleic acid encoding the protein of SEQ ID NO: 399 or a fragment thereof may be administered to an individual. Alternatively, a formulation that increases the protein activity of SEQ ID NO: 399 may be administered to the individual. Such formulations include the protein of SEQ ID NO: 399 or SEQ ID NO: 399
Can be identified by contacting cells or preparations containing the protein with a test agent and assaying whether the test agent increases the activity of the protein. For example, the test drug is
It can be a compound or polypeptide or peptide.

Alternatively, the protein activity of SEQ ID NO: 399 can be reduced by administering to the individual a formulation that interferes with such activity. A formulation that interferes with the activity of the protein of SEQ ID NO: 399 is to contact a cell or preparation containing the protein of SEQ ID NO: 399 or the protein of SEQ ID NO: 399 with a test drug and determining whether the test drug reduces protein activity. Can be identified by For example, the formulation may be a compound, polypeptide or peptide, antibody, or nucleic acid such as an antisense nucleic acid or a triple helix forming nucleic acid. In one embodiment, the present invention selectively identifies a tissue, preferably brain, based on the protein level of SEQ ID NO: 399 in a sample, or if two or more sources of said sample are possible. The present invention relates to a method and a composition using the protein of the present invention or a part thereof as a marker protein for identifying the protein. For example, the protein of SEQ ID NO: 399 or fragments thereof can be used to raise antibodies using techniques known to those of skill in the art, including those described herein. Such tissue-specific antibodies can then identify tissues of unknown origin, such as differentiated tumor tissues that have metastasized to forensic samples, different body sites, etc., or use immunochemistry to distinguish different tissue types in tissue sections. Can be used for In such methods, a tissue sample is contacted with a detectably labeled antibody under conditions that promote antibody binding. The level of antibody binding to the test sample is measured and compared to the level of binding to control cells from brain or non-brain tissue to determine if the test sample is brain derived. Alternatively, the protein level of SEQ ID NO: 399 in the test sample may be measured by determining the RNA level encoding the protein of SEQ ID NO: 399 in the test sample. RNA levels can be measured using nucleic acid arrays or by methods such as in situ hybridization, Northern blot, dot blot or other methods known to those of skill in the art. If desired, an amplification reaction such as a PCR reaction may be performed on the nucleic acid sample prior to analysis. The RNA level in the test sample is compared to that of control cells from brain or non-brain tissue to determine if the test sample is from brain. In other embodiments, antibodies to the proteins of the invention or portions thereof can be used to detect, enrich, or purify cells expressing the protein of SEQ ID NO: 399, including techniques known to those of skill in the art. For example, an antibody to the protein of SEQ ID NO: 399 or a fragment thereof can be immobilized on a solid support such as a chromatography matrix. A preparation comprising cells expressing the protein of SEQ ID NO: 399 is contacted with the antibody under conditions that promote binding to the antibody. The cells are released from the support by washing the support and then contacting the support with a drug that dissociates the cells from the antibody.

In another embodiment of the invention, the protein of SEQ ID NO: 399 or fragments thereof can be used to diagnose disorders associated with altered expression of the protein of SEQ ID NO: 399. In certain embodiments, the protein of SEQ ID NO: 399 or fragments thereof can be used in cancer diagnosis. In such methods, the protein level of SEQ ID NO: 399 in diseased individuals is measured using methods such as those described herein. The protein level of SEQ ID NO: 399 in diseased individuals is compared to the level in healthy individuals. For example, elevated protein levels of SEQ ID NO: 399 as compared to a healthy individual suggests that the sick individual may have cancer, or may be predictively diagnosed to have cancer in the future. In another embodiment, the invention provides: (i) transforming a host cell with an expression vector comprising a polynucleotide encoding SEQ ID NO: 399; and (ii) culturing the transformed host cell expressing the protein, And (iii) a method for preparing the PAP protein of SEQ ID NO: 399, which comprises the step of isolating the protein. The present invention further comprises a method for dephosphorylation of a substrate, comprising contacting the substrate with an effective amount of the isolated protein of SEQ ID NO: 399 or a fragment thereof so that the protein catalyzes the dephosphorylation of the substrate. Regarding Further, it is provided that the method is realized in vitro and comprises the step of isolating the dephosphorylated substrate. Furthermore, the method can occur in vivo and is carried out by administering the protein (or part thereof) of the invention to a mammal in need thereof. <Proteins of SEQ ID NOs: 258 and 262 (internal designation numbers 110-007-1-0-C7-CS, 16-
055-1-0-A3-CS)> The protein of SEQ ID NO: 258 is encoded by the cDNA of SEQ ID NO: 17.
Therefore, it is understood that all features and uses of the polypeptide of SEQ ID NO: 258 described throughout this application also relate to the polypeptide encoded by the human cDNA which is clone 110-007-1-0-C7-CS. Will. In addition, all features and uses of the nucleic acid of SEQ ID NO: 17 described throughout this application also include clone 110-007-1-0-
It will be appreciated that it relates to the nucleic acid encoded by the human cDNA which is C7-CS. The protein of SEQ ID NO: 258 is 2 of GENESEQP accession numbers W96745 and W41056.
It shows homology to two high affinity IgE receptor-like proteins (IGER), the disclosures of which are incorporated herein by reference in their entireties. The protein of SEQ ID NO: 258 is expressed in liver and testis. SEQ ID NO: 262, encoded by SEQ ID NO: 21, is a polymorphic variant of the protein of SEQ ID NO: 258 and shares all of the potential functions and uses described herein. This protein and cDNA
Shares all features and uses and products of the 116-055-1-0-A3-CS clone.

Like these two high-affinity IgE receptor-like proteins, the proteins of the invention have a transmembrane domain of 20 amino acids with amino acids 53-73, 79-99, 121.
~ 141 and 158-178, respectively. The protein of SEQ ID NO: 258 is
It penetrates the plasma membrane four times, forming two small extracellular loops with both N- and C-termini in the cytoplasm. Furthermore, the protein of the present invention contains a signal peptide (a cleavage site at position 21). The predicted structure of the protein of SEQ ID NO: 258 is that of this protein, FcεRIβ and CDC.
It shows relationships with 20 antigens and provides evidence for a family of four transmembrane proteins. Conservation of amino acids in all three proteins is highest in the four transmembrane domains above. Although there are greater differences at the hydrophilic amino and carboxyl termini, multiple amino acids are conserved within these regions, such as the presence of four prolines at the amino termini of all three proteins. ing. In addition, there are two cysteine residues (positions 147 and 156) in the second extracellular domain between TM3 and TM4. This suggests the presence of intermolecular or intramolecular disulfite bonds in this domain in all three proteins. FcεRI is part of a tetrameric receptor complex consisting of an α chain, a β chain and two γ chains (Kinet et al., Proc Natl. Acad. Sci. USA, 15: 6483-6487 (1988), Is incorporated herein by reference in its entirety). Together, they mediate interactions with IgE-binding antigens to reach dramatic cellular responses such as massive degranulation of mast cells. The β subunit is a four-transmembrane protein with both amino and carboxyl termini in the cytoplasm. Chromosome mapping was performed using the cDNA of SEQ ID NO: 17 as the position of the CD20 gene on chromosome 11.
Located on q12. However, mouse FcεRIβ and Ly-44 (in mice
CD20 equivalents) are both located at the same site on mouse chromosome 19 (Tede
r, TF et al., J. Immunol. 141: 4388-4394 (1988), Clark EA and Lane, JL.
Annu. Rev. Immunol. 9: 97-127 (1991), the disclosures of which are incorporated herein by reference in their entirety). Thus, these three genes appear to have originated and evolved at the same location, further supporting that they are members of the same family of related proteins.

Based on the above information, the protein of SEQ ID NO: 258 has the high affinity immunoglobulin E
It seems to be a receptor-like protein. Atopic diseases, including allergies, asthma, atopic dermatitis (or eczema) and allergic rhinitis are commonly defined as impaired immunoglobulin E (IgE) response to common antigens such as pollen or house dust mite. This is often detected by positive skin tests for elevated total serum IgE levels, antigen-specific IgE responses or common allergens. In principle, atopy is the response of IgE to antigen exposure and interaction of IgE with its high affinity Fc receptor, FcεRI, a receptor on its mast cells, and its subsequent ligand-receptor binding. Due to dysregulation somewhere in the pathway that begins with cellular activation by Ravetch, Nature
Genetics, 7: 117-118 (1994), the disclosure of which is incorporated by reference in its entirety). Accordingly, the invention provides a protein of SEQ ID NO: 258, at least 5 of which
, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150 or 200 contiguous amino acid fragments, or fragments having the desired biological activity are sequenced. No. 258 protein can be administered to an individual who desires an increase or decrease in activity. In particular, the protein of SEQ ID NO: 258 or a fragment thereof is administered to an individual in whom it is desired to control the IgE response range. In such a method, the protein of SEQ ID NO: 258 or fragment thereof may be administered directly to the individual, or a nucleic acid encoding the protein of SEQ ID NO: 258 or fragment thereof may be administered to the individual. Alternatively, a formulation that increases the protein activity of SEQ ID NO: 258 may be administered to the individual. Such formulations can be identified by contacting the protein of SEQ ID NO: 258 or cells or preparations containing the protein of SEQ ID NO: 258 with a test agent and analyzing whether the test agent increases the activity of the protein. For example, the test agent can be a chemical compound or polypeptide or peptide. In addition, the protein of SEQ ID NO: 258 or fragments thereof can be used to identify genes or polypeptides involved in IgE response or atopic disease. In particular, the binding partners of the protein of SEQ ID NO: 258, or the genes encoding such binding partners, can be identified using various techniques known to those of skill in the art, including those described herein.

In addition, the protein activity of SEQ ID NO: 258 or the polynucleotide encoding the protein of SEQ ID NO: 258 can be used to diagnose hereditary atopy. In particular, the level in the test individual of the protein of SEQ ID NO: 258 is determined by the method as described herein, and compared to the level of the healthy individual and the individual suffering from hereditary atopy, the test individual is heritable. It is possible to determine whether the patient has atopy or is at risk of having the disease. Alternatively, by obtaining and analyzing a nucleic acid sample from a test individual, R encoding the protein of SEQ ID NO: 258 associated with hereditary atopy
It is possible to determine the level of NA, or whether it contains a mutation in the gene encoding the protein of SEQ ID NO: 258 associated with hereditary atopy. For example, a nucleic acid sample from a test individual is contacted with a nucleic acid probe containing a nucleic acid that encodes the protein of SEQ ID NO: 258 or a fragment thereof, the RNA level of which, or the individual has a mutation associated with hereditary atopy. Can be determined. The probe may be DNA, including cDNA or genomic DNA, or the probe may be
It may be RNA. Any of the techniques known to those of skill in the art can be used for these diagnostic methods, including the methods as described herein. For example, the presence of mutations associated with hereditary atopy is characterized by PCR, sequencing or mini-sequencing (Biochem. Biophys. Res. Comm., 182: 50, as described in Yamamoto et al.
7 (1992), the disclosure of which is incorporated herein by reference in its entirety), but is not limited to these and can be determined by methods known in the art. The protein of SEQ ID NO: 258 can be used to characterize the expression of FcεRI and the induction of specific functions of FcεRIβ. Therefore, the protein of the present invention is, for example, Fcε
It may be useful for the design of drugs that block or suppress the induction or activity of RI, thereby treating atopic disease. In particular, test agents that block or suppress induction or activity can be identified by the methods described herein. In other embodiments, the protein of SEQ ID NO: 258 can be used to produce antibodies, such as monoclonal antibodies, using methods known in the art, including those described herein. Antibodies can be used to block or mimic ligand binding to the proteins of the invention or to receptors including, but not limited to, FcεRI and other receptors. The antibody can be used to isolate the protein of SEQ ID NO: 258, or cells expressing the protein of SEQ ID NO: 258, in a manner as described herein. For example, the antibody can be used to determine the presence of cells containing the protein of SEQ ID NO: 258, including but not limited to hematopoietic cells. For example, this method comprises contacting a sample with an antibody under conditions sufficient for the antibody to bind to the protein of SEQ ID NO: 258, and binding antibody by methods known in the art, including those described herein. Consists of detecting the presence of.

In one embodiment, the invention selectively identifies a tissue, preferably a tissue such as liver and testis, based on the protein level of SEQ ID NO: 258 in the sample, or the source of said sample is 2 Methods and compositions that use the proteins of the invention or portions thereof as marker proteins to identify them when considered more than one. For example, the protein of SEQ ID NO: 258 or fragments thereof can be used to raise antibodies using techniques known to those of skill in the art, including those described herein. Such tissue-specific antibodies can then identify tissues of unknown origin, such as differentiated tumor tissues that have metastasized to forensic samples, different body sites, etc., or use immunochemistry to classify different tissue types in tissue sections. Can be used for In such methods, a tissue sample is contacted with a detectably labeled antibody under conditions that promote antibody binding. Measuring the level of antibody binding to the test sample,
The level of binding to control cells from the liver or testis, or tissue other than liver or testis, is compared to determine if the test sample is from the liver or testis. Alternatively,
The protein level of SEQ ID NO: 258 in the test sample may be measured by determining the RNA level encoding the protein of SEQ ID NO: 258 in the test sample. RNA levels can be measured using methods such as nucleic acid arrays or in situ hybridization, Northern blot, dot blot or other methods known to those of skill in the art. If desired, an amplification reaction such as a PCR reaction may be performed on the nucleic acid sample prior to analysis. The RNA level in the test sample is compared to the RNA level in control cells from the liver or testis, or tissue other than the liver or testis to determine if the test sample is from the liver or testis. <Protein of SEQ ID NO: 279 (Internal Designation No. 160-58-3-0-H3-CS)> The protein of SEQ ID NO: 279 is encoded by the cDNA of SEQ ID NO: 38. Accordingly, all of the features and uses of the polypeptide of SEQ ID NO: 279 described throughout this application also relate to the polypeptide encoded by the nucleic acid contained in clone 160-58-3-0-H3-CS. Will be understood. In addition, all of the features and uses of the nucleic acid of SEQ ID NO: 38 described elsewhere in this application also include
It will be appreciated that it relates to the nucleic acid contained in clone 160-58-3-0-H3-CS. The protein of SEQ ID NO: 279 is encoded by a 1330 nucleotide nucleic acid having an ORF that results in a 267 amino acid protein between 198 and 998 nucleotides. This protein is a polymorphic variant of the sequence of proenkephalin A precursors (including Met- and Leu-enkephalin) (SP: P01210). It has a signal peptide spanning 24 amino acids and two signature motifs, a vertebrate endogenous opioid neuropeptide and an endogenous opioid neuropeptide precursor. PSORT predicts extracellular localization involving the cell wall (66.7%).
The protein of SEQ ID NO: 279 is mainly distributed in the fetal brain, although it was also expressed in other tissues (see below). A polymorphic variant was detected at amino acid position 75 (
E-> D which is a conservative amino acid change). Cleavage of signal peptide (amino acids 47-267
220 amino acids), the protein still contains a polymorphic variant at amino acid position 29. This does not change the sequence of the different enkephalins that occur after cleavage of the precursor protein. In addition, the polymorphism is 25 amino acids away from the first cleavage site on the amino terminus. Therefore, it can be said that the secondary structure of the actual cleavage site is unlikely to change.

International Patent Application Publication WO9606863-A1, whose disclosure is incorporated herein by reference in its entirety, discloses a protein showing high homology with the protein of SEQ ID NO: 279. Therefore, the protein of SEQ ID NO: 279 appears to be enkephalin. Met- and Leu-enkephalin compete with and mimic the effects of opiate drugs. These two pentapeptides with potent opiate agonist activity in bioassay systems were first identified by Hughes et al.
Nature, 258, 577-580, 1975). The natural ligands of the opiate receptor, which differ only in the COOH-terminal amino acid, reflect the fact that their source is the brain, Met- and L
It was named eu-enkephalin. Peptides containing these sequences are called opium or opioid peptides. Enkephalin is widely distributed throughout the central nervous system in the enkephalin-acting neural network, and is also present in the peripheral nervous system such as the autonomic ganglia. Predominantly contextual data are, for example, modulation of pain perception, endogenous opioids on mood and behavior, learning and memory, response to stress, diverse neuroendocrine functions, immune regulation and cardiovascular and respiratory functions. Suggesting a widespread involvement in. Met-enkephalin enhances the immune response in patients with cancer or AIDS. This allows the binding of opoid receptors present in peripherally inflamed tissue to mediate the analgesic effect. Includes antibody production, NK cell activity against tumor and viral infections, macrophage and polymorphonuclear leukocyte function, graft rejection, and mitogen-stimulated lymphocyte proliferation after exogenous administration of different enkephalins , Multiple immune functions are affected. The effect may be bidirectional, with the same immune function being amplified at low concentrations and suppressed at high concentrations. Therefore, enkephalin is a modulator of the immune response. These opioid neuropeptides are released by the post-translational proteolytic process of precursor proteins. These multivalent precursor proteins (polyproteins) follow a conserved region of about 50 residues, a variable length region and a sequence of diverse neuropeptides,
It consists of a signal sequence. Preproenkephalin A (PENK gene) is processed to produce the following peptides, including Met-enkephalin (6 copies are present, 2 of which are extended) and Leu-enkephalin: 1 -24 Signal peptide 100-104 Peptide Met-enkephalin 1 107-111 Peptide Met-enkephalin 2 136-140 Peptide Met-enkephalin 3 186-193 Peptide Met-enkephalin-arg-gly-leu 210-214 Peptide Met-enkephalin 4 230 -234 peptide Leu-enkephalin 261-267 peptide Met-enkephalin-arg-phe The N-terminal conserved regions of these precursors probably contain 6 cysteines involved in disulfide bonds. This region may also be important for neuropeptide processing.

Precursor proteins have the potential to be specifically cleaved into multiple extended enkephalin- and non-enkephalin-containing peptides of almost unknown function. However, in some cases extended enkephalin-containing peptides have been shown to have enhanced opiate activity. Another peptide produces enkelytin, which exhibits antibacterial activity (see below). Proenkephalin is present predominantly independent of free enkephalin peptide in cell types, including some tissues and astrocytes (Melner et al., EMBO.
J, 9, 791-796, 1990; Spruce et al., EMBO J 9, 1787-1795, 1990, the disclosure of which is incorporated herein by reference in its entirety), released untreated from these cells. (Batter et al., Brain Res. 563, 28-32, 1991, the disclosure of which is incorporated by reference herein in its entirety) is increasing in number. There is evidence that in some cases the treated enzyme is co-released with untreated precursors, suggesting that extracellular cleavage occurs (Vilijn et al., J. Neurochem 53, 1).
487-1493, 1989). Even though biological activity is signaled by the binding of small peptide products to cell surface receptors, regulation of this activity may be mediated by precursors, and the untreated precursors may be And may have an additional role in the cell. This protein is, first and foremost, striatum, as well as neuroendocrine tissue,
It is described to exist in various brain regions of the pituitary gland and the adrenal gland. This protein is expressed on ConA-stimulated CD4 T lymphocytes, CD4 thymocytes, B lymphocytes and various immune cells including T cell lines, macrophages and mast cells. Expression in the reproductive system, heart and many developing tissues during pregnancy and early postpartum has been reported. Therefore, it has been postulated that these peptides play some role in cell or tissue growth and differentiation. For example, endogenous enkephalins induced in thymocytes function to modulate their expression and suppress the proliferation of activated thymocytes. Enkephalin peptides are abundant in the adrenal medulla and can be released by neurotransmitters specific for that tissue. Enkephalin has been found to be abundant in human pheochromocytoma, a tumor derived from the adrenal medulla. This tumor RNA contains high levels of enkephalin mRNA sequences, as demonstrated by cell-free translation studies.

Enkephalins are classified as delta, kappa and mu and function as opiate receptors. A study by Lord et al. (Nature, 267, 495-499, 1977) compared the activities of morphine and enkephalin in a bioassay system and found that enkephalin binds predominantly to the delta receptor. Subsequent studies have shown that these receptors and the OBCAM (Sch
ofield et al., EMBO J 8, 489-495, 1989, the disclosure of which is incorporated herein by reference in its entirety) and the somatostatin receptor (International Patent Application Publication No. WO 96/0).
No. 6863, the publication of which is incorporated herein by reference in its entirety), and has revealed homology with other receptor families. This can explain the former reported opioid binding properties. By the homology with the latter opiate receptor,
Expected to bind opioid receptor ligands. Recognition of opioid peptides by other non-opiate-related receptors implies that these peptides perform other yet unknown functions. In addition, enkephalin is involved in apoptosis. Apoptosis is a morphologically unique controlled process of cell death that modulates the process of cell production by mitosis. A molecular link was established between the regulation of cell production and cell clearance, including the roles of c-myc and p53 in the pathways that mediate apoptotic cell death. All mammalian cells, in the absence of continuous signaling from adjacent cells,
It is proposed to be programmed to die as a default. However, in response to the level of genetic damage associated with common environmental insults, obtaining a life-prolonging effect that prevents single cells from activating the suicide program may lead to the survival of potential oncogenic mutations. Since it becomes advantageous, it can theoretically be an event that causes carcinogenesis. Alternatively, improper activation of survival pathways can lead to the abolition of the endogenous death program, promoting early and late tumorigenesis. A particularly potent oncogenic pathway is probably due to the promotion and tolerance of genetic damage, which may have helped the cell overcome its extracellular survival signal need. About 50
% Human tumors have normal p53 function. Therefore, there is a need to identify additional pathways or molecules that inappropriately block apoptosis in human tumors. Opioid-like molecules may be involved in such pathways.

There are reports in the literature that pathways involving opioid-like molecules are involved in controlling the balance between cell death and survival. For example, morphine suppresses cell survival in the developing cerebellum (Hauser et al., Exp. Neurol, 130, 95-105.
, 1994, the disclosure of which is incorporated herein by reference in its entirety), induces apoptosis in thymocytes (Fuchs and Pruett, J. Pharmacol. Exp. T.
her. 266, 417-423, 1993, the entire disclosure of which is incorporated herein by reference). In a series of experiments (WO 96/06863), proenkephalin and / or its proteolytic products were: a) deficient in exogenous survival factors, b
) When the exogenous survival factor is unrestricted, it has been discovered to act as an extracellular and / or cell surface membrane binding factor that modulates cell survival of transformed cells after genotoxic injury and / or stress. It was The single or multiple receptors most likely present on the cell surface and to which these single or multiple factors bind are
Related to, or possibly identical to, one or more members of the opioid receptor family. Opioid-like receptor types or subtypes can mediate survival or death. The single or multiple receptors that mediate death appear to be coupled to those that mediate survival. The natural ligand of the receptor can be the product of the opioid precursor gene, but it is also possible that the natural ligand contains cytokines that mimic these effects. Tumor cells are more sensitive than untransformed cells to antagonize opioid-like receptor-mediated survival and to stimulate opioid-like molecular receptor-mediated death.
Induction of cell cycle arrest sensitizes tumor cells to these manipulations. (The enhanced sensitivity of tumor cells to these manipulations is triggered by their co-occurrence in the cell cycle. Cytoplasmic proenkephalins and / or their proteolytic products act as general repressors of apoptosis. Formulations capable of antagonizing cytoplasmic proenkephalin when combined with a suitable internalizing agent are therefore useful in inducing apoptosis in non-transformed as well as transformed cells, especially when combined with sublethal doses of apoptosis-inducing agents. Can be.

Apoptosis mediated by cytoplasmic proenkephalins is activated at high cell densities, mainly by non-dispersive factors. Therefore, in order to reduce exogenous survival signaling and simulate low densities, formulations were prepared with neutralizing antibodies against eg integrins (such as the 23C6 antibody-Bates et al., J. Cell Biol. 125 403-415,
1994) can synergistically enhance the inhibition of the above-mentioned proenkephalin or its product. Proenkephalin targeted to the cell nucleus induces apoptotic death that is suppressed by overexpression of large T antigen and is mediated at least in part by p53. Tumors that retain wild-type p53 function are therefore specific targets for elevated levels of proenkephalin or its derivatives in the nucleus or induction of apoptosis by agents that mimic the function of nuclear proenkephalin or its derivatives. Therefore, the protein of SEQ ID NO: 279, a fragment thereof, or SEQ ID NO: 2.
Nucleic acids encoding 79 proteins or fragments thereof can be used to modulate biochemical pathways involving the opioid peptide precursor gene. In some embodiments, antibodies or other formulations that reduce the level or activity of the protein of SEQ ID NO: 279 or fragments thereof can be used to induce apoptosis in cells. Preferably, the formulation neutralizes the protein of SEQ ID NO: 279 or a proteolytic derivative thereof and increases, activates or mimics the level of nuclear proenkephalin, or is associated with or identical to the delta and or kappa opioid receptors. Acts as an antagonist to the receptor for. In some embodiments, the formulation may be a neutralizing monoclonal antibody against the protein of SEQ ID NO: 279 or fragments thereof. Furthermore, the formulations may be fragments or allelic forms of these antibodies. In addition, the formulation includes a cytoplasmic anchor,
Alternatively, it may include a nuclear localization signal. In some embodiments, the formulation is capable of modulating the biochemical pathways of cells in which apoptosis is induced by involving the product of the opioid peptide precursor gene. The formulation can be used to induce apoptosis in lens cells after treatment of cancer or after cataract surgery. In some embodiments, the formulation promotes apoptosis of proliferating cells with less or no effect on normal mature cell types. The formulation may be administered in combination with a genotoxic or cell cycle arresting agent. Alternatively, the formulation may be complexed with chemotherapeutic agents, irradiation agents or cell cycle arresting agents (tuning agents).

Accordingly, the present invention provides a means of inducing apoptosis in a cell, including altering the biological pathways of the cell in which the product of the opioid precursor gene is involved so that apoptosis is induced. . Altered routes may be effected by administration of the appropriate formulation. In particular, the invention provides a formulation for use in inducing apoptosis in cells, which formulation comprises: a formulation capable of neutralizing proenkephalin or a proteolytic derivative thereof; nuclear proenkephalin and / or its Formulations that increase levels of, or activate or mimic, derivatives, related to delta opioid receptors, or acting as antagonists at the same single or multiple receptors, or related to kappa opioid receptors Or a formulation that acts as an agonist at the same single or multiple receptors. A subset of such formulations are those that are capable of neutralizing proenkephalin or proteolytic derivatives, formulations that act as antagonists at or at the same single or multiple receptors as the delta opioid receptor, or kappa opioid receptors. It is a formulation that acts as an agonist at a single or receptor associated with or identical to the body. In some embodiments, the formulation may be administered to the cell surface such that the survival effect of extracellular and / or cell surface membrane-bound proenkephalin or a proteolytic derivative thereof is neutralized, resulting in Makes cells become apoptotic in nature ,. Alternatively, the formulation capable of neutralizing proenkephalin or its proteolytic derivative may be bound to the internalizing peptide and the cytoplasmic anchor. Such a composition remains in the cytoplasm of the cell and antagonizes cytoplasmic proenkephalin and / or its proteolytic products, thus neutralizing the anti-apoptotic effect of these molecules. In addition, enkephalin has antibacterial activity. During treatment with proenkephalin-A, maturation in adrenal medulla chromaffin cells begins with the removal of the carboxy terminus (proenkephalin-A derived peptide or PEAP209-239) (Y. Goumon, K. Lugardon
, B. Kieffer et al., J. Biol. Chem. 273: 29847-29856, 1998, the disclosures of which are incorporated herein by reference in their entireties). The enkeritin peptide was identified as the equivalent of the diphosphorylated PEAP209-237, Staphylococcus aureus, and Micrococcus luteus and Giant bacterium (0.2-0.4 μM range)
It has antibacterial activity including other Gram-positive bacteria. Gram-negative bacteria (E. coli strain
D22, D31, 663 and T13773) neither have the ability to influence the proliferation nor the hemolytic activity. The activity of this peptide is specific, and the shortened version of this peptide (209-220, 224-2)
37, 230-237, 233-237), or non-phosphorylated PEAP209-239 exhibits only a slight bacterial growth inhibitory activity.

Bovine periarthritis abscess fluid contains different forms of PEAP (72-237 / 239; 80-237 / 239), as confirmed by immunoreactivity and sequence analysis. These peptides have activity against M. luteus, but less than enchelitin (0.2 μM for 5). These PE
AP constitutes a pool of precursors to be processed in order to supply activated enkelitin during infection. Similarly, the presence of PEAP with a molecular weight corresponding to that of PEAP209-237 was also detected. In addition, PEAP (PEAP202-238 and PEAP206-2
37) was detected in wound fluid, including post-Caesarean section abscesses in the subcutaneous layer and abscesses induced by subcutaneous injection of Freund's complete adjuvant. Therefore,
These peptides are found in wound fluid in the presence of other known antimicrobial peptides (defensin (
coexist with defensins) and bactenecins). These concentrations were in the same concentration range (0.5-1 μM) as the level showing activity in vitro. Furthermore P
EAP was also detected in the secretions of human polymorphonuclear neutrophils. PEAP209-230 and enkelitin are secreted by cultured chromaffin cells after stimulation. This suggests that these two peptides are released with catecholamines in stress situations and thus play an important role in the defense mechanism. The co-release of met-enkephalin and enkelitin may represent a unified neuroimmune protective response to stress situations that may be associated with infection. This could provide a highly beneficial survival strategy much earlier in the proinflammatory process. Thus, this protein may play an important role in host defense against bacterial infections, especially those involving Gram-positive bacteria. Due to their non-specific activity towards these membranes, antimicrobial peptides have cytotoxic activity and may play some role not only in antimicrobial defense, but also in inflammatory processes, and possibly wound repair. The protein of SEQ ID NO: 279, peptides derived from its cleavage, or fragments thereof may be used as an antimicrobial agent, topically, in creams / ointments / solutions, presoaked dressings, or skin patches. Alternatively, the protein of SEQ ID NO: 279, a peptide derived from its cleavage, or a fragment thereof can be used for injection (for intravenous, subcutaneous or intraperitoneal injection). This is useful for wound repair, burn healing and postoperative recovery management.

Alternatively, the protein of SEQ ID NO: 279, a peptide derived from its cleavage, or a fragment thereof is used for surface cleaning, such as in the home (cooking room, bathroom) or workplace (tabletop, telephone, computer keyboard and mouse). You may mix with the sterilizing liquid. Another application is as an additive for mouthwash or pop-up wet tissues. Changes in enkephalin levels can result in psychological illness. Konig et al. (Nat
ure, 383, 535-538, 1996, the disclosure of which is incorporated herein by reference in its entirety), used genetic methods to study the role of the mammalian opioid system. The authors disrupted the preproenkephalin gene using homologous recombination in embryonic stem cells to produce enkephalin-deficient mice.
The enk-/-mutant animals are healthy, fertile, care for their offspring, but show considerable behavioral abnormalities. Mice with the enk-/-genotype show more anxiety and males show more aggressive aggression. Mutant animals show striking differences in supraspinal response to painful stimuli compared to control animals, but not spinal responses. However, these enk − / − mice show normal stress-induced analgesia. Thus, enkephalins modulate the response to painful stimuli. Therefore, genetic factors can contribute significantly to the pain experience. This study clearly demonstrates the importance of enkephalin in pain perception, anxiety and aggression. Interestingly, the PENK gene is localized at 8q23-q24, the same location where genes associated with epilepsy and spastic paraplegia, cerebral dysfunction-related disorders exist. Therefore, the SEQ ID NO: 279 protein or fragment thereof is associated with a psychological disorder,
In particular, it can be used to treat distortions in pain perception, disorders that include aggression, or anxiety. This means drug addiction, different types of phobia, panic attacks, schizophrenia,
It may include bipolar, anorexia nervosa, chronic pain disorders, post-traumatic events, post-surgical pain management. Accordingly, the invention provides a protein of SEQ ID NO: 279, at least 5 of this protein.
, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150 or 200 contiguous amino acid fragments, or for the treatment or amelioration of a disease in an individual , Including the use of fragments having the desired biological activity. For example, the disease may be cancer, a disease caused by increased or decreased cell proliferation, a bacterial infection, a disease caused by an abnormal immune response, a psychological disease, or any of the diseases listed above. In such embodiments, the protein of SEQ ID NO: 279, or a fragment thereof, is administered to an individual where it is desired to increase or decrease any of the activities of the protein of SEQ ID NO: 279. The protein of SEQ ID NO: 279 or fragment thereof may be administered directly to the individual, or the nucleic acid encoding the protein of SEQ ID NO: 279 or fragment thereof may be administered to the individual. Alternatively, SEQ ID NO: 279
An agent that increases the protein activity of the may be administered to the individual. Such formulations are
It can be identified by contacting the protein of SEQ ID NO: 279 or a cell or preparation containing the protein of SEQ ID NO: 279 with a test agent and analyzing whether the test agent increases the activity of the protein. For example, the test agent can be a compound or polypeptide or peptide.

Alternatively, the protein activity of SEQ ID NO: 279 may be reduced by administering to the individual a formulation that prevents such activity. A formulation that interferes with the activity of the protein of SEQ ID NO: 279 is by contacting a cell or preparation containing the protein of SEQ ID NO: 279 or the protein of SEQ ID NO: 279 with a test agent and determining whether the test agent reduces protein activity. Can be identified. For example, the formulation can be a compound, polypeptide or peptide, antibody, or nucleic acid such as an antisense nucleic acid or a triple helix forming nucleic acid. In one embodiment, the invention selectively identifies the origin of the sample, e.g., fetal brain, based on the protein level of SEQ ID NO: 279 in the sample, or if two or more origins are considered, those The present invention relates to methods and compositions using the protein of the present invention or a part thereof as a marker protein for identifying For example, the protein of SEQ ID NO: 279 or fragments thereof can be used to raise antibodies using techniques known to those of skill in the art, including those described herein. Such antibodies can then be used to identify tissues of unknown origin, such as differentiated tumor tissues that have metastasized to forensic samples, different body sites, etc., or to classify different tissue types in tissue sections using immunochemistry. it can. In such a method, the sample is
Contact is made with the antibody labeled for detection under conditions that promote antibody binding.
The level of antibody that binds to the test sample is measured and compared to the level of binding to fetal brain or control cells derived from tissues other than fetal brain to determine if the test sample is derived from fetal brain. Alternatively, the protein level of SEQ ID NO: 279 in the test sample may be measured by determining the RNA level encoding the protein of SEQ ID NO: 279 in the test sample. RNA levels can be measured using methods such as nucleic acid arrays or in situ hybridization, Northern blots, dot blots, or other methods known to those of skill in the art. If desired, an amplification reaction such as a PCR reaction may be performed on the nucleic acid sample prior to analysis. The RNA level in the test sample is compared to that of control cells derived from fetal brain or a tissue other than fetal brain to determine if the test sample is derived from fetal brain. In other embodiments, antibodies to the protein of the invention or portions thereof can be used to detect, enrich, or purify cells expressing the protein of SEQ ID NO: 279, including techniques known to those of skill in the art. For example, an antibody against the protein of SEQ ID NO: 279 or a fragment thereof can be immobilized on a solid support such as a chromatography matrix. A preparation comprising cells expressing the protein of SEQ ID NO: 279 is contacted with the antibody under conditions that promote binding to the antibody. The cells are released from the support by washing the support and then contacting the support with a drug that dissociates the cells from the antibody.

In another embodiment of the invention, the protein of SEQ ID NO: 279 or a fragment thereof can be used to diagnose disorders associated with altered expression of the protein of SEQ ID NO: 279. In such a method, the protein level of SEQ ID NO: 279 in a diseased individual is measured using a method as described herein. The protein level of SEQ ID NO: 279 in a diseased individual is compared to the level in a healthy individual to determine if the individual has a protein level of SEQ ID NO: 279 associated with the disease. <Protein of SEQ ID NO: 293 (internal designation number 181-16-1-0-G7-CS)> The protein of SEQ ID NO: 293 is a protein encoded by HSPC163 (Gene Bank Accession No. AF161512), gene number 93 ( PCT / US99 / 17130) and human cornichon protein show high homology with TGAM77. SEQ ID NO: 293 is overexpressed in cancer prostate, fetal brain and fetal kidney. The HSPC163 gene is one of 300 cDNAs obtained from the CD34 + hematopoietic stem / progenitor cell (HSPC) library (derived from cord blood and adult bone marrow). Furthermore, HSPC163 is NB4
(Granulocytic), HL60 (granulocytic), U937 (mononuclear), K562 (red / megakaryocytic),
And Jurkat (T lymphoid) were identified in five hematopoietic cell lines. These cell lines represent different hematopoietic cell lineages. The polypeptide of Gene No. 93 was determined to have a transmembrane domain and a short cytoplasmic tail. Based on these characteristics, the protein product of Gene No. 93 appears to share structural similarity with type IIIa membrane proteins. This gene is expressed primarily in activated T cells and to a lesser extent in endometrial tumors, T cell helper II cells, microvascular endothelial cells, cyclohexamide-treated Raji cells, and umbilical vein endothelial cells. Has been done. The expression pattern of Gene No. 93 indicates a role in controlling proliferation, survival, differentiation, and / or activation of hematopoietic cell lineages, including blood stem cells. Gene products appear to be involved in the control of cytokine products, antigen presentation, and other immune processes, suggesting that they help strengthen the immune system. The translation product of this gene has a high degree of homology with human TGAM77 and mouse cornion protein. TGAM77 was identified as a gene involved in the early stage of T cell activation in response to alloantigen. Twenty-four hours after T cell alloactivation, TGAM77 RNA expression is significantly increased. TGAM77 has been designated as a T cell growth-related molecule. TGAM7 is the human homologue of the Drosophila cornion (cni) protein.

It has been demonstrated that cornification is involved in carefully modulated signaling events during Drosophila oogenesis and establishes an asymmetric pattern in oocytes as a prerequisite for correct embryogenesis. . Cornion signaling functions in concert with two other proteins. The function of all three genes in the EGF-like signaling pathway appears to govern the formation of a correctly polarized microtubule cytoskeleton, which is associated with oocyte polarization, asymmetric nuclear migration, and embryonic development. It is believed to be the basis for the correct spatial localization of other signaling molecules essential for differentiation. The present invention provides an amino acid sequence of SEQ ID NO: 293 and a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 293. In one embodiment, SEQ ID NO: 293
Polypeptide is compatible with the corresponding polypeptide encoded by the human cDNA clone 181-16-1-0-G7-CS. The invention also includes bioactive fragments of the protein of SEQ ID NO: 293, and polynucleotide sequences encoding these bioactive fragments. “Bioactive fragment” is defined as a peptide or polypeptide fragment of SEQ ID NO: 293 that has at least one of the biological functions of a full-length protein (eg, the ability to stimulate T cell proliferation). The invention also provides variants of the protein of SEQ ID NO: 293. These variants have at least about 80%, preferably at least about 90%, and most preferably at least about 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 293. Variants according to the invention may also have SEQ ID NO: 293, such as the biological functions described above.
Has at least one functional or structural characteristic of. The invention also provides bioactive fragments of variant proteins. Unless otherwise stated, the methods disclosed herein can be performed using SEQ ID NO: 293 or variants thereof.
Similarly, the methods of the invention can be practiced with a biological fragment of SEQ ID NO: 293, or a variant of said biologically active fragment. Due to the redundancy of the genetic code, various DNA sequences can encode the amino acid sequence of SEQ ID NO: 293. It is well within the skill of those skilled in the art to create these alternative DNA sequences that encode proteins having identical or substantially identical amino acid sequences. These mutant DNA sequences are therefore within the scope of the present invention. As used herein, a “substantially identical” sequence means a sequence having amino acid substitutions, deletions, additions or insertions that does not substantially affect biological activity. Fragments having one or more characteristic biological activities of SEQ ID NO: 293 are also included in this definition.

A “recombinant nucleotide variant” is an alternative polynucleotide that encodes a particular protein. They can be synthesized, for example, by taking advantage of the "redundancy" of the genetic code. Diverse codon substitutions, such as silent changes that produce specific restriction sites or codon usage-specific mutations, optimize cloning into plasmids or viral vectors, or expression in particular prokaryotic or eukaryotic host systems, respectively. Can be introduced for. SEQ ID NO: 293 protein and variants thereof can be used to raise antibodies by methods known in the art. The aforementioned antibodies can be monoclonal or polyclonal. Antibodies can also be synthesized by known methods against fragments of SEQ ID NO: 293 as well as variants of SEQ ID NO: 293. The invention also provides an antibody that specifically binds to the bioactive fragment of SEQ ID NO: 293 or a variant of the SEQ ID NO: 293. The invention also provides immunoassays used to screen, monitor, or diagnose prostate cancer. Methods of screening, diagnosing, identifying, or monitoring the process of prostate cancer are known to those of skill in the art. In this aspect of the invention, the biological sample (eg, blood, serum, tissue, or biopsy tissue sample) is SEQ ID NO: 293, SEQ ID NO: 2.
An immunoassay is provided that is contacted with an antibody that specifically binds an immunogenic fragment of 93, or a bioactive fragment of SEQ ID NO: 293. The immune complex formed in the contacting step is then detected using a suitable labeled detection reagent. The expression level of SEQ ID NO: 293 in the biological sample tested is compared to the control / normal level commonly measured for that population. Alternatively, a method for screening, monitoring, or diagnosing prostate cancer can be performed using SEQ ID NO: 293, or a fragment of SEQ ID NO: 293, as well as a nucleic acid encoding SEQ ID NO: 293, or a fragment of SEQ ID NO: 293. it can.
In one embodiment, the polypeptide can be used as a standard / control in the immunoassays described above. In another embodiment, SEQ ID NO: 293 is used to identify a biological sample containing SEQ ID NO: 293 (eg, blood, serum, tissue, or biopsy tissue sample).
Nucleic acid encoding, or a fragment of SEQ ID NO: 293 is used in hybridization assays known to those of skill in the art. SEQ ID NO: 293 in the biological sample tested
The expression level of is compared to control / normal levels commonly measured for that population.

In another embodiment, SEQ ID NO: 293 and the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 293 are used to identify immune disorders involving activated T cells using standard hybridization assays. Or it can be used to diagnose. In another aspect, the invention provides a method of immunostimulating a mammal. In this aspect of the invention, SEQ ID NO: 293 and / or a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 293 is introduced into T cells according to known methods. The T cells are then activated by activation with an antigen to induce the mammalian immune system. In other embodiments, autologous T cells are obtained from the individual. SEQ ID NO: 293, a biologically active fragment thereof, and / or a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 293 or the biologically active fragment of SEQ ID NO: 293 is introduced into autologous T cells according to known methods. The T cells proliferate and are reintroduced into the individual from whom the T cells were obtained. For example, the entire text of which is incorporated herein by reference, U.S. Pat.
See 192,537 and 5,766,920. In another embodiment of the invention, the polynucleotides and polypeptides encoding SEQ ID NO: 293 are proliferation of stem cells that are restricted precursors of various blood lineages, and
It can be used for the differentiation and / or proliferation of various cell types. In this aspect of the invention, the polynucleotides and polypeptides encoding SEQ ID NO: 293 are introduced into cells and the cells are cultured. These methods can be carried out according to methods known to those skilled in the art. <Protein of SEQ ID NO: 316 (Internal Designation No. 188-45-1-0-D9-CS)> The protein of SEQ ID NO: 316 is encoded by the cDNA of SEQ ID NO: 75.
It is therefore understood that all of the features and uses of the polypeptide of SEQ ID NO: 316 described throughout this application also relate to the polypeptide encoded by the nucleic acid contained in clone 188-45-1-0-D9-CS. Will Moreover, all of the features and uses of the nucleic acid of SEQ ID NO: 75 described throughout this application also apply to clone 188-45.
It will be understood that it pertains to the nucleic acid contained in 1-0-D9-CS. The protein of SEQ ID NO: 316 is expressed in brain and has three transmembrane segments located between positions 6 and 26, between positions 7 and 93, or between amino acids 139 and 159, and the sequence FAAFCYMLSLVLC / AA. With a signal peptide containing. Accordingly, one embodiment of the invention is a polypeptide that includes one or more transmembrane segments and / or signal peptides.

The protein of SEQ ID NO: 316 is a member of the cornition protein family. This protein is the Drosophila melanogaster ecological protein
48% identity, as well as TGAM77 Human Cornition homolog (Genbank accession number AF1
No. 04398, the entire text of which is incorporated herein by reference) and 67%
HCornichon, which is a bone marrow secretory protein (International Patent Application Publication No. WO / 99339
No. 79, the disclosure of which is hereby incorporated by reference in its entirety) with 67% identity, a human secretory protein encoded by gene 24 (International Patent Application Publication No.
WO / 9910363, the disclosure of which is incorporated herein by reference in its entirety)
It has 67% identity and 67% identity with the protein product of the mouse cnih gene. However, this protein has a high degree of homology to the mouse cornilion-like protein (Genbank Accession No. AB006191, the disclosure of which is incorporated herein by reference in its entirety), which is a product of the mouse cnil gene. % Identity. The protein of SEQ ID NO: 316 is encoded by the reproductive gene 95, human secretory protein (GSP: Y76218, International Patent Application Publication No. WO / 9958660, the disclosure of which is incorporated by reference in its entirety into the present specification. Have a high level of identity, and are presumably polymorphic variants of gene 95. The high rate of sequence conservation among members of this family indicates that they are under strong selective pressure and are probably involved in important cellular functions. The product of the Drosophila ecological (cni) gene is involved in the signaling process that is required for the formation of both Drosophila, anterior-posterior and dorsoventral patterns during Drosophila embryogenesis (Cell, 1995, 81: 967- 978). Mutations in the cornion prevent the formation of correctly polarized microtubule cytoskeletons in oocytes. Cni signaling functions by modulating with the other two proteins. A single epidermal growth factor (EGF) that is structurally most similar to vertebrate TGFα
Glucene, a protein secreted from oocytes containing motifs, is believed to be a ligand for torpedo, a homologue of the Drosophila epidermal growth factor receptor (DER) expressed by follicular epithelium. The function of all three genes in the EGF-like signaling pathway appears to direct the formation of a correctly polarized microtubule cytoskeleton, which formation involves oocyte polarization, asymmetric nuclear migration, and embryonic development. It is believed to be the basis for correct spatial localization of other signaling molecules essential for differentiation. TGAM77, one of the human homologues of the cornion, is an alloactivated T
Differentially expressed in cells (Bioch. Biophys. Acta 1999, 1449: 203
-210, the full text of which is incorporated herein by reference). TGAM77 is required for directional localization of T cell activation signaling molecules, as it is known that the microtubule cytoskeleton is involved in spatial polarization in T cell activation signaling events. It is believed that it may function in the protein tyrosine phosphatase pathway.

The protein of SEQ ID NO: 316 was detected in brain tissue and gene 95 (GSP: Y76218, International Patent Application Publication No. WO / 9958660, the publication of which is incorporated herein by reference), was identified in pediatric brain. It is expressed in tissues, endometrial tumor tissues and frontal cortex tissues. Furthermore, ESTs corresponding to this gene have been detected in lung tissue, sex cell tumors and cutaneous melanoma. This is similar to the expression pattern of the mouse cnil gene found in whole day 6.5 embryos, day 11.5 limb buds, day 13.5 whole embryos, adult lung and brain (Dev. Genes Evol. , 1999, 209: 120-125, the disclosure of which is incorporated herein by reference in its entirety). A polynucleotide that encodes the protein of SEQ ID NO: 316 or a fragment thereof, and a polypeptide that comprises the protein of SEQ ID NO: 316 or a fragment thereof, is of a single or multiple tissues or single or multiple cell types present in the biological sample It is useful for reagents for specific identification and diagnosis of diseases and disorders including, but not limited to, endometrial tumors and neurological and developmental diseases and / or injuries. Similarly, antibodies to the protein of SEQ ID NO: 316, or fragments thereof, and antibodies to these polypeptides are useful in providing immunological probes for the identification of single or multiple tissues or cell types. In some disorders involving the above tissues or cells, particularly neural and reproductive organs, certain tissues or cell types (eg, neural, reproductive, cancerous and injured tissues) or body fluids (eg, lymph, serum, Plasma, urine, amniotic fluid, synovial fluid and spinal fluid) or other tissue or cell samples taken from individuals with such disorders, with standard gene expression levels (ie expression in normal tissues or fluids from individuals without the disorder). Level), the expression of this gene at significantly elevated or decreased levels can be detected by conventional means. Tissue distribution in pediatric brain tissue and adult brain tissue, and homology with the Cornion protein, the polynucleotide encoding the protein of SEQ ID NO: 316 or a fragment thereof, and a polypeptide comprising the protein of SEQ ID NO: 316 or a fragment thereof, It has been shown to be useful in detecting and / or diagnosing nerve and developmental injury. Tissue distribution, these polynucleotides and polypeptides, Alzheimer's disease, Parkinson's disease, Huntington's disease, Tourette's syndrome, schizophrenia, mania, dementia, paranoia, obsessive-compulsive disorder, learning disability, ALS, psychosis, Detection of neurodegenerative disease states and behavioral disorders such as autism and behavioral changes including disorders in eating, sleep patterns, balance, and perception
It has been shown to be useful in treatment. In addition, the gene or gene product may play a role in the treatment and / or detection of developmental disorders associated with the developing fetus, or sexually-related disorders.

Increased expression in the brain of the protein of SEQ ID NO: 316 suggests that it is involved in neuronal cell survival, synapse formation, membrane conductivity, neural differentiation, etc. Such engagement can affect a number of processes such as learning and cognition. Alternatively, the tissue distribution in endometrial injured tissue, germ cell tumors and cutaneous melanoma may be determined by translation products of this gene, such as endometrial tumor and / or reproductive disorders, and expression of this gene. It is useful for the detection and / or treatment of tumors in the above tissues. In addition to its use as a nutritional supplement, the protein of SEQ ID NO: 316 also determines bioactivity, produces antibodies, isolates cognate ligands or receptors as tissue markers, or their mutual interactions. It can also be used to identify formulations that modulate the effect. The protein of SEQ ID NO: 316 or fragments thereof, and antibodies to the protein can be used as tumor markers and / or immunotherapy targets in the tissues listed above. The gene encoding the protein of SEQ ID NO: 316 appears to be on chromosome 11. Accordingly, the gene encoding the protein of polynucleotide SEQ ID NO: 316 or a fragment thereof is useful as a marker in chromosome 11 linkage analysis. Accordingly, the invention provides a protein of SEQ ID NO: 316, at least 5 of that protein.
, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150, or 200 contiguous amino acid fragments, or treating or ameliorating a disease in an individual In order to include the use of fragments with the desired biological activity. For example, the disease may be an abnormality in development, signaling pathways, microtubule organization, neuronal survival, synapse formation, conductance, neural differentiation, or cancer, or an abnormality of any of the functions listed above. In such embodiments, the protein of SEQ ID NO: 316, or a fragment thereof, is administered to an individual in whom it is desired to increase or decrease any of the activities of the protein of SEQ ID NO: 316. The protein of SEQ ID NO: 316 or fragment thereof may be administered directly to the individual, or the nucleic acid encoding the protein of SEQ ID NO: 316 or fragment thereof may be administered to the individual. Alternatively, a formulation that increases the protein activity of SEQ ID NO: 316 may be administered to the individual. Such formulations include the protein of SEQ ID NO: 316 or SEQ ID NO: 316.
Can be identified by contacting cells or preparations containing the protein with a test agent and assaying whether the test agent increases the activity of the protein. For example, the test drug is
It can be a compound or polypeptide or peptide.

Alternatively, the protein activity of SEQ ID NO: 316 may be reduced by administering to the individual a formulation that prevents such activity. A formulation that interferes with the activity of the protein of SEQ ID NO: 316 is by contacting a cell or preparation containing the protein of SEQ ID NO: 316 or the protein of SEQ ID NO: 316 with a test agent and determining whether the test agent reduces protein activity. Can be identified. For example, the formulation can be a compound, polypeptide or peptide, antibody, or nucleic acid such as an antisense nucleic acid or a triple helix forming nucleic acid. In one embodiment, the invention selectively identifies the origin of a sample, such as, for example, the brain, based on the protein level of SEQ ID NO: 316 in the sample, or if two or more origins of the sample are considered. The present invention relates to methods and compositions using the protein of the present invention or a part thereof as a marker protein for identifying For example, the protein of SEQ ID NO: 316 or fragments thereof can be used to raise antibodies using techniques known to those of skill in the art, including those described herein. Such antibodies can then be used to identify tissues of unknown origin, such as differentiated tumor tissues that have metastasized to forensic samples, different body sites, etc., or to distinguish different tissue types in tissue cross sections using immunochemistry. Can be used. In such methods, a sample is contacted with a detectably labeled antibody under conditions that promote antibody binding. The level of antibody binding to the test sample is measured and compared to the level of binding to control cells from brain or non-brain tissue to determine if the test sample is brain derived. Alternatively, the protein level of SEQ ID NO: 326 in the test sample is compared to SEQ ID NO: 3 in the test sample.
It may be measured by determining the RNA levels encoding 26 proteins. R
NA levels can be measured using methods such as nucleic acid arrays or in situ hybridization, Northern blots, dot blots, or other methods known to those of skill in the art. If desired, an amplification reaction such as a PCR reaction may be performed on the nucleic acid sample prior to analysis. The RNA level in the test sample is compared to that of control cells from brain or non-brain tissue to determine if the test sample is from brain. In other embodiments, antibodies to the proteins of the invention or portions thereof can be used to detect, enrich, or purify cells expressing the protein of SEQ ID NO: 316, including techniques known to those of skill in the art. For example, an antibody against the protein of SEQ ID NO: 316 or a fragment thereof can be immobilized on a solid support such as a chromatography matrix. A preparation comprising cells expressing the protein of SEQ ID NO: 316 is contacted with the antibody under conditions that promote binding to the antibody. The cells are released from the support by washing the support and then contacting the support with a drug that dissociates the cells from the antibody.

In another embodiment of the invention, the protein of SEQ ID NO: 316 or a fragment thereof can be used to diagnose a disease associated with modified expression of the protein of SEQ ID NO: 316. In such methods, the protein level of SEQ ID NO: 316 in diseased individuals is measured using methods such as those described herein and compared to levels in healthy individuals. The protein level of SEQ ID NO: 316 in a diseased individual is compared to the level in a healthy individual to determine if the individual has a protein level of SEQ ID NO: 316 associated with the disease. <Protein of SEQ ID NO: 255 (106-037-1-0-E9-CS.cor)> The protein of SEQ ID NO: 255 encoded by the cDNA of SEQ ID NO: 14 is strongly expressed in the liver and testis and Lactate dehydrogenase-A protein (LDH-
It shows considerable homology to the A or M chains (Chung FZ et al., Biochem. J. 231:
537-541 (1985); SWISSPROT accession number P00338). Furthermore, the protein of SEQ ID NO: 255 shows homology to many vertebrate lactate dehydrogenase A. The protein of 381 amino acids long SEQ ID NO: 255 exhibits a Prosite motif corresponding to lactate dehydrogenase at positions 71 to 380. In addition, the LGEHGDS active site (where H is the active site residue)
It is present in the proteins of the invention (positions 239-245). The protein of the present invention further contains 50 N-terminal additional amino acids not found in other lactate dehydrogenase A proteins. This N-terminal extension contains a signal peptide (cleavage site at position 34 of the protein of the invention) that can reveal protein export to the extracellular domain, cell membrane export, or specific subcellular localization. Alternatively, the initiation start codon may be at position 26 or 50 of the protein of SEQ ID NO: 255. Lactate dehydrogenase (LDH), along with the hydrogen acceptor NAD +, is an enzyme that dehydrogenates lactic acid to pyruvate. Exists (Abad-Zap
atero C. et al., J. Mol. Biol. 198: 445-467 (1987)). In vertebrates, type M (LDH-A) is mainly detected in muscle tissue, type H (LDH-B) is detected in heart muscle, and type X (only in mammalian and avian sperm) ( It is known that there are three types of LDH isozymes, LDH-C). LD in the eye lens of birds and crocodiles
HB acts as a structural protein and is known as epsilon-crystallin (Hendriks W. et al., Proc. Natl. Acad. Sci. USA 85: 7114-7118.
(1988)).

LDH has been used extensively for a number of purposes in the field of clinical trial reagents. Used as a binding enzyme to measure the enzymatic activity of various aminotransferases, such as alanine aminotransferase (ALT), which is ultimately detected by UV spectroscopy of the pyruvate produced. ing. Such applications of LDH have been widely adopted as clinical tests because aminotransferases show high activity in the liver, heart, kidney and the like, and significantly increase in serum with various diseases. In addition, LDH is also used as a binding enzyme to help measure levels of substrates such as urea, which LDH converts such substances to pyruvate, which is detectable by UV spectroscopy. Because it promotes. Lactate dehydrogenase is widely used as a marker for heart disease and other diseases. For example, LD-1 levels are elevated in the presence of other diseases such as myocardial infarction and leukemia. Lactate dehydrogenase levels begin to rise 24-48 hours after coronary artery closure, peak at 3-6 days, and return to normal at 8-14 days. In addition, the level of LD-1 rises 10-12 hours after acute myocardial infarction, peaks 2-3 days, and reaches approximately 7-1.
Return to normal in 0 days. Therefore, measuring lactate dehydrogenase levels allows a retrospective diagnosis of myocardial infarction. Furthermore, the amount of LD-2 in the blood is usually higher than that of LD-1, but in the case of patients with acute myocardial infarction, LD-1 is present in a larger amount than LD-2.
This "reversal ratio" usually returns to normal in 7-10 days. The LD-1 level of the increased reversal ratio is
It has a sensitivity and specificity of about 75% to 90% for the detection of acute myocardial infarction. Furthermore, elevated LDH levels have also been used as a prognostic indicator for cancers such as small cell lung carcinoma. In particular, elevated LDH levels indicate that the course of these diseases is unwelcome (Kawahara et al. (1997) Jpn J Clin Oncol199.
7 Jun; 27 (3): 158-65). Furthermore, LDH expression in cells has been shown to be induced by interleukin-1 alpha, a major cytokine associated with inflammation, for example (Neha
r et al., (1998) Biol Reprod Dec; 59 (6): 1425-32. The islet beta cells have low expression levels of lactate dehydrogenase and high glyceryl phosphate dehydrogenase activity. The effect of acute overexpression of the skeletal muscle isoform of lactate dehydrogenase (LDH) -A on glucose metabolism and insulin secretion in these cells was studied by Ainscow EK et al. (Diabetes 2000 Jul; 49 (7)). : 1149)
. The results of these studies indicate that overexpression of LDH activity interferes with normal glucose metabolism and insulin secretion in islet beta cells, and thus is a direct cause of insulinotropic defects in some forms of type 2 diabetes. It can be shown that Furthermore, these results support the notion that glucose-derived pyruvate metabolism in mitochondria is essential for the secretion of glucose-stimulating insulin in beta cells. Other data show that overexpression of lactate dehydrogenase A
It has been shown to attenuate glucose-induced insulin secretion in a stable MIN-6 beta cell line that normally expresses low levels of L-lactate dehydrogenase (Zhao C,
Rutter GA FEBS Lett. 1998 Jul 3; 430 (3): 213-6). Therefore, low LDH activity appears to be important in beta-cell glucose sensitivity.

In addition, analysis of LDH isozyme patterns in CSF fluid was shown to help assess CNS involvement in patients with hematological malignancies (Lossos IS et al., Canc.
er.2000 Apr 1; 88 (7): 1599-604). The protein of SEQ ID NO: 255 is believed to be lactate dehydrogenase, probably of LDH-A or M subtype. The activity of this protein can be measured by a standard enzyme activity detection method for lactate dehydrogenase, including that involving the detection of pyruvate, which is a product of LDH-catalyzed enzymatic reaction, by ultraviolet rays. In one embodiment, the polynucleotides and polypeptides of the invention are used to detect testis and liver tissues, and cells derived from these tissues. For example, the nucleic acids and proteins of the invention can be prepared using methods known to those of skill in the art,
It can be isotopically or chemically labeled and used as a probe in Northern blots, Far Western blots, and in situ hybridization experiments. The ability to specifically detect kidney cells is useful, for example, in determining the history of tumor cells, and in identifying specific cell types and tissues for histological analysis. In other embodiments, the protein can be used in any of a variety of clinical assays involving the LDH enzyme. For example, the protein can be used as a conjugated enzyme to measure the enzymatic activity of various aminotransferases, such as alanine aminotransferase (ALT), detected by ultraviolet spectrophotometric analysis of produced pyruvate.
Such an assay has clinically significant usefulness because aminotransferase is an enzyme that exhibits high activity in liver, heart, kidney, etc., and significantly increases in serum with various diseases. . In addition, the proteins of the invention can be used as a binding enzyme to help measure levels of substrates such as urea, which enzyme converts such substances into pyruvate, which can be detected by UV spectroscopy. This is because it promotes the conversion of In other embodiments, the protein can be used to identify cosmetic ingredient compositions. Specifically, a lactate dehydrogenase enhancer can be included in the cosmetic composition to activate keratinocyte proliferation and collagen synthesis in skin tissue. Inhibitors include pyruvic acid, acetic acid, acetoacetic acid, beta-hydroxybutyric acid, pathway metabolites of the Krebs cycle, aliphatic saturated or unsaturated fatty acids containing 8 to 26 carbon atoms, containing 22 to 34 carbon atoms Can be combined with other active ingredients such as omega-hydroxy acid, glutamic acid, glutamine, valine, alanine, leucine, and mixtures thereof (eg, US Pat. No. 5,855).
(See No. 3,742, the full text of which is incorporated herein by reference)
.

In another embodiment, the invention provides a method of treating or preventing cancer, eg, by inhibiting lactate dehydrogenase activity in cells, preferably cancer cells of a patient. Expression or activity of lactate dehydrogenase includes a number of preparations including, but not limited to, antibodies, antisense molecules, ribozymes, and heterologous molecules that suppress expression or activity of lactate dehydrogenase in cancer cells of patients. Can be suppressed by using any of the above. In one embodiment, a lactate dehydrogenase obtained from primate,
Alternatively, an anti-lactate dehydrogenase antibody obtained as a result of parenteral administration of primate lactate dehydrogenase from a mammal to the mammal is parenterally administered to a human cancer patient. In addition, antibodies derived from the proteins of the invention or portions thereof can be used to suppress cancer cell development, as described in US Pat. No. 4,620,972. Analysis of LDH isozyme patterns in CSF fluid has been shown to help assess CNS involvement in patients with hematological malignancies (Lossos IS et al. Cancer. 2000.
Apr 1; 88 (7): 1599-604). Therefore, in another embodiment, the protein of SEQ ID NO: 255 can further be used in the development of an assay to monitor LDH isozyme activity in CSF fluid, thereby improving the sensitivity of CSF cytology. This assay may be derived, for example, from the method described by Short S. et al. (J Biol Chem. 2000 Apr 28; 275 (17): 12963-9). In another embodiment, the protein of SEQ ID NO: 255 has been used to detect and / or treat insulinotropic defects in some forms of type 2 diabetes. For example, various lines of evidence indicate that LDH overexpression is involved in some forms of diabetes. Thus, detection of elevated levels of LDH in a patient, for example, in a patient's islets of Langerhans, can be used as an indicator that the patient has or is at risk of developing diabetes. Similarly, LD in these cells
Methods of inhibiting H expression or activity using, for example, antibodies, antisense sequences, or heterologous compounds that inhibit LDH expression or activity can be used to treat or prevent diabetes. In other embodiments, the proteins of the invention can be used to remove endogenous pyruvate in cells in vitro or in vivo. In other embodiments, expression of the protein is used as a marker of activity of interleukin 1, eg IL-1alpha, in cells or patients. Specifically, since LDH expression was shown to be induced by IL-1alpha, expression of this protein or elevated expression can be used as a marker for the action of IL-1 on cells. Since IL-1 is implicated in a number of physiological processes including inflammation, and more specifically in adverse processes such as arthritis and autoimmune disorders, the protein is present in the presence or absence of such disorders. Serves as a marker for predisposition.

In another embodiment, the protein is used to detect heart disease and other diseases in patients. For example, LDH levels are known to increase after myocardial infarction and other heart diseases. Thus, detection of elevated levels of the proteins of the invention, alone or in light of the levels of other proteins such as other LDH isozymes, can be used as an indicator of other diseases including heart attack or leukemia. LDH levels can be measured in any tissue or biological sample, including but not limited to serum, using standard methods, including but not limited to immunoassays and LDH enzyme activity assays. Can be detected. In another embodiment, the protein is used to determine the prognosis of a number of diseases, including cancers such as small cell lung carcinoma. For example, the level of the protein of the present invention is detected in the serum of a patient suffering from cancer, and the level of protein expression or activity is detected to be lower as compared with the prognosis of a patient having a normal level of protein activity or expression. If so, it indicates a poor prognosis for the patient. <Proteins of SEQ ID NOs: 243 and 253 (internal designation numbers 105-016-1-0-D3-CS and 105-095-2-0-G11-CS)> 331 amino acids in length encoded by the cDNA of SEQ ID NO: 2 The protein of SEQ ID NO: 243 was detected in the prostate and fetal brain and is a secreted human protein (Genseq accession number
Y59685) and is homologous. Furthermore, this protein is higher than the putative glycerophosphate diester phosphodiesterase (GP-PDE) MIR16 (membrane interaction protein of RGS16) protein (SPTREMBLNEW SPTREMBL SWISSPROT accession number AAF65234) encoded by the cDNA of GENPEPT GENPEPTNEW accession number AF212862. It shows homology and indeed the proteins of the invention are possible variants of the MIR16 protein. Furthermore, a BLAST search using the amino acid sequence of SEQ ID NO: 243 shows that the protein of the present invention is homologous to GP-PDE of Escherichia coli (SWISSPROT accession numbers P09394 and P10908) and Haemophilus influenzae (SWISSPROT accession number Q06282). The protein of SEQ ID NO: 243 has two transmembrane segment candidates of amino acids 7-27 and 258-278,
The putative signal peptide of amino acids 19-24 is presented. Finally, the protein of the invention has two putative N-glycosylation sites: asparagine residues 168 and 198 positions (Zheng et al., Proc. Natl. Acad. Sci. 97: 3999-4004 (2000)). .

The cDNA of SEQ ID NO: 2 has GENPEPT GENPEPTNE at its extended 5'and 3'ends.
Unlike the cDNA of W Accession No. AF212862, it differs from the cDNA of SEQ ID NO: 12 in polymorphism and alternative splicing. The MIR16 (membrane interaction protein of RGS16) protein homologous to the protein of the present invention was identified in the yeast 2 hybrid screen of the pituitary cell cDNA library fed with the RGS16 (G protein signal transduction regulator) protein (Zheng.
Proc. Natl. Acad. Sci. 97: 3999-4004 (1999)). And Sasaki, J. Bacte.
riol. 175: 4569-4571 (1993); Zheng et al., ibid.). Surprisingly, the H. influenzae GP-PDE (also called protein D), which is 67% identical to the periplasmic GP-PDE of E. coli, has an affinity for human immune globin D (Janson et al., Infect. 62: 4848). -854 (19
94)). By sequence alignment, N of MIR16 immediately following the putative signal peptide
The terminal region (amino acids 70-150) is clearly conserved (40-61% similar), suggesting that it contains residues that are important for catalytic activity, ie the catalytic site. To do. GP-PDE hydrolyzes deacetylated phospholipids GP such as glycerophosphocholine (GPC) and glycerophosphoethanolamine to sn-glycerol-3-phosphate (G3P) and the corresponding alcohols (Zheng et al., Ibid). MI
The putative enzymatic activity of R16 and its interaction with RGS16 suggest that it plays an important role in lipid metabolism and G protein signaling.
MIR16 mRNA is transcribed in a significant proportion in heart, liver, kidney, testis and brain, as evidenced by Northern blot experiments. The expression of MIR16 found in the brain is consistent with the above expression of the protein of the invention in fetal brain. The proteins of SEQ ID NOs: 243 and 253, or parts thereof, are members of the glycerophosphate diester phosphodiesterase protein family and interact with the RGS16 protein and thus play an important role in the therapy of lipid metabolism and G protein signaling. Preferred polypeptides of the present invention are 7-2
A polypeptide comprising the amino acids of SEQ ID NO: 243 at positions 7, 19-24 and 258-278. Other preferred polypeptides of the present invention are fragments of SEQ ID NO: 243 or 253, which have any of the biological activities described herein. Further preferred polypeptides are those containing asparagine residues at positions 168 and / or 198.

The present invention is first directed to specific cell types using the cDNA of SEQ ID NO: 2 or 12, or a portion thereof, and the protein of SEQ ID NO: 243 or 253 of the present invention or a portion thereof,
Preferably it relates to methods and compositions for identifying specific cell types from the prostate or fetal brain. For example, the nucleic acids and proteins of the invention are labeled with isotopes or chemically according to methods known to those of skill in the art and further probed in Northern blots, far western blots, and in situ hybridization detection experiments. Can be used as The ability to detect specific cell types is useful, for example, in determining the history of tumor cells and in identifying cells and tissues for histological research purposes. A number of in vitro assays can be used to detect activity of the protein of SEQ ID NO: 243 or 253, such as in vitro screening for modulators of protein activity. Preferably, it encodes a protein of the invention
The cDNA is cloned into a prokaryotic expression vector according to methods known to those skilled in the art. That is, the GP-PDE activity of the recombinant protein was analyzed by the coupled spectrophotometric method described by Larson and coworkers and used by Cameron and coworkers (Larson et al., J. Biol. Chem. 258: 5426-5432 (1983); Camer
on et al., Infect. Immun. 66: 5763-5770 (1998)). Such enzymatic activity can be measured in vitro in the presence of the modulating drug. Another embodiment of the invention relates to a method of using the protein or a part thereof for the purification of human immunoglobin D or its specific binding. Some of the immune globin (Ig) -bound bacterial cell wall proteins have been isolated and / or cloned in the last 20 years. The best characterized of these is that it binds to Staphylococcus aureus protein A (human IgG1, 2 and 4 subclasses, numerous mammalian IgGs and, in some cases, human Igs A, M, E classes). ), And Group G beta hemolytic streptococcal protein G, which binds to all human IgG subclasses and also exhibits a broader binding spectrum for IgG than protein A. IgD does not bind to protein A or protein G. Therefore, identifying new proteins with the ability to bind IgD is of great interest, which allows the separation and purification of the proteins. What is more, IgD binding proteins can be used in immunoprecipitation procedures with IgD, as is commonly done with proteins A and G in the case of IgG. Binding and purification of IgD using the proteins of the invention can be performed using any of a number of methods, for example, using recombinant DNA molecules, the proteins of the invention, or portions thereof, with other proteins. Fusion proteins or polypeptides that are combined may be produced and performed. The fusion product, containing a protein of the invention or a portion thereof, is then covalently or by some other means attached to a protein, carbohydrate or matrix (gold, "Sephadex" particles, polymer surface, etc.). can do. Such a complex is very useful for immobilization of IgD and continuous batch-wise immunoprecipitation. Protein D (GP-PDE) Haemophilus influenzae and I
A similar assay for binding to gD is described in US Pat. No. 6,025,484.

In another embodiment, the invention provides a protein of the invention, or a portion thereof, a deacylated phospholipid (GP) such as glycerophosphocholine (GPC) and glycerophosphoethanolamine, sn-glycerol-. Methods and compositions for use as GP-PDE enzymes that hydrolyze to 3-phosphate (G3P) and corresponding alcohols. First of all, due to this enzymatic activity associated with a specific phospholipase D class, the proteins of the invention are very useful for studying biomembranes and phosphos and their phospholipid components. Furthermore, since glycerophospholipids are a major element of the lipid bilayer, removal of these hydrophilic moieties using the GP-PDE activity of the proteins of the invention probably led to permeation of eukaryotic cell membranes and, consequently, permeation. Will change gender. Such changes may improve the efficiency of eukaryotic gene transfer in vitro or in vivo. Generally, in such embodiments, the purified protein of SEQ ID NO: 243 or 253 is administered to a cell, but the purified protein of the invention can be purified using any of the several existing methods, for example, The cDNA encoding the protein can be inserted into a prokaryotic expression vector and purified using any of the techniques known to those skilled in the art. The recombinant protein produced and purified in the prokaryotic system is then added to the in vitro culture of eukaryotic cells before or during gene transfer. Furthermore, the recombinant proteins of the invention, most notably in the case of gene therapy,
It can also be used to increase the efficiency of in vivo cell gene transfer. For example, masses of mass are often resistant to gene transfer, and the proteins of the invention are likely to induce cytotoxic genes (such as pro-apoptotic genes) in solid solid tumors.
Or it could possibly provide an efficient way to facilitate the introduction of anti-tumor drugs. Yet another embodiment of the proteins of the invention relates to methods and compositions for diagnosing, treating, and preventing disorders associated with excess glutamate signaling in the brain. As described above, the MIR16 protein physically interacts with the RGS16 protein (G protein signaling regulator 16). Many hormonal receptors use heterotrimeric G proteins for signal transduction after ligand binding (see Neer, Cell 80: 249-257 (1995) for a review). Among these receptors are the metabotropic glutamate receptors (mGluRs). Like the proteins of the invention, these receptors expressed in the brain are a novel family of cloned G protein-coupled receptors (Schoepp and Conn, Trends Pharmacol. Sci. 14:
13-20 (1993)). Endogenous glutamate may contribute to the brain damage that occurs acutely after epilepsy, cerebral ischemia or traumatic brain injury by activating mGluR1 receptors (and including NMDA and AMPA receptors). It may further contribute to chronic neurodegeneration in disorders such as amyotrophic lateral sclerosis and Huntington's disease (Meldrum, J. Nu.
tr. 130 (4S Suppl): 1007S-1015S (2000)).

Accordingly, the present invention provides for the diagnosis, treatment, and treatment of disorders associated with excess glutamate signaling in the brain, with the cDNA of SEQ ID NO: 2 or 12, or a portion thereof, and the protein of SEQ ID NO: 243 or 253, or a portion thereof. It relates to methods and compositions used to prevent. Specifically, the activity or expression level of the protein is the level of glutamate signaling, or brain damage associated with glutamate signaling involved in epilepsy, cerebral ischemia, traumatic brain injury, ALS, or Huntington's disease,
Alternatively, it can be correlated with any of the physiological processes or diseases or disorders associated with other G proteins. When the expression or activity level of the protein is in the positive direction thereof and correlates with the presence of the above-mentioned signal transduction, disease, or medical condition, any method for detecting the protein expression or activity can be used. Signal transduction, disease, medical condition can be detected. These methods of protein expression or activity include Northern blotting, far western blotting, and in situ hybridization when an elevated level of the protein, protein activity, or nucleic acid of the present invention indicates the presence of a disease, medical condition, or signal transduction. Including experiments. Furthermore, with compounds, such as antibodies, antisense molecules, ribozymes, dominant negatives of proteins, that suppress the expression or activity of the protein, or any heterologous molecule that suppresses the activity or expression of the protein, such A disease or disorder can be treated or prevented, or such signaling pathways can be suppressed. Alternatively, if the expression or activity of the protein of the invention is associated in its negative direction with a signal transduction pathway, disease or disorder, then a reduced level of protein expression or activity is expressed to indicate the presence of the disease, disorder or pathway. Detection can be used. Further, in such cases, the nucleic acid encoding the protein,
Any compound that increases the activity or level of the protein, such as the protein or a heterologous compound that causes an increase in the expression or activity of the protein, can be used to treat or prevent a disease or disorder, or inhibit a pathway. <Protein of SEQ ID NO: 386 (Internal Designation Number 105-037-4-O-H12-CS)> The protein of SEQ ID NO: 386 encoded by the cDNA of SEQ ID NO: 145 is strongly expressed in fetal brain and uterus. . The protein of SEQ ID NO: 386, which is 207 amino acids long, exhibits a pfam SPRY domain at positions 85-205.

The SPRY domain has been detected in numerous cells and multiple proteins involved in developmental processes. For example, the Midline-1 / FXY family of proteins
It is said to be involved in human diseases such as Opitz's syndrome, which is a congenital disorder that is associated with microtubules and characterized by numerous developmental abnormalities (for example, Ca
inarca et al. (1999) Hum Mol Genet 8 (8): 1387-96). Furthermore, the cytoplasmic Marenostrin / Pyrin protein has been demonstrated to be responsible for familial Mediterranean fever, an autosomal recessive disorder characterized by fever and serositis (Nat Genet 19
97 Sep; 17 (1): 25-31). Other SPRY proteins include SplA, a Staphylococcus aureus serine protease, and butyrophilin, a major milk protein. SP
Another family of proteins known to contain the RY domain is the ryanodine receptor (RyRs). The ryanodine receptor plays an important role in Ca2 + signaling in muscle and non-muscle cells by releasing Ca2 + from intracellular stores. For example, these receptors are excitatory-contractile-coupled (EC), which occurs in a specialized region where the sarcoplasmic reticulum (SR) containing the ryanodine receptor and plasma membrane / transverse tubule system form a bond.
Coupling). Furthermore, RyRs appear to play some role in maintaining the structural integrity of SR / T tubule junctions. However, because RyR interacts with other macromolecules at the junction of the triad, it clearly cannot perform the necessary functions associated with EC coupling. For example, this calmodulin and
Two small proteins of FKBP12 are believed to modulate RyR at the junction of this triad. Mammalian tissues are thought to express three different RyR isoforms,
These isoforms consist of four 560-kDa (RyR polypeptides) and four 12-
It contains the kDa (FK506 binding protein) subunit. These large protein complexes conduct monovalent and divalent cations and appear to be capable of multiple interactions with other molecules. The subunits of the protein complex are small endogenous diffuse effector molecules, including Ca2 +, Mg2 +, adenine nucleotides, sulfhydryl modifying reagents (glutathione, NO, and NO adducts) and lipid intermediates, and protein kinases and phosphatases. , Calmodulin, immunophilin (FK506 binding protein) and, in skeletal muscle, proteins such as the dihydropyridine receptor. The skeletal muscle RyR is the tissue's predominant calcium release channel and is the most intensively studied of the three genetic isoforms ever detected in mammals. The other two RyR isoforms, often referred to as "heart" or "brain", are complex in their actual cellular and tissue distribution.

Due to their multiple ligand interactions, the ryanodine receptor constitutes an important and potentially abundant pharmacological target for the regulation of cell function. Ca2 + release channel activity is modulated by a number of endogenous effectors, including Ca2 +, ATP, Mg2 +, and calmodulin. In addition, numerous exogenous effectors, including caffeine, local anesthesia, and polyamines also fluctuate channel activity.
For example, tetracaine, procaine, benzocaine, and lidocaine suppress the release of calcium from SR. They appear to interact with single or multiple specific sites located on the RYR, affecting both ryanodine binding and single channel activity (Shoshan-Barmatz et al., 1993; J. Membr. Biol. ; 133
171-181). The importance of intracellular calcium as a second messenger in cell signaling processes is clear. Changes in intracellular Ca2 + homeostasis include secretion, contraction-relaxation,
There are significant effects on multiple cell functions including motility, metabolism, protein synthesis, modification and folding, gene expression, cell cycle progression and apoptosis. The major sources of cytosolic calcium are intracellular storage sites located in the endoplasmic reticulum, or within the sarcoplasmic reticulum (SR) in muscle. Since cellular Ca2 + treatment is an important factor in the regulation of neuronal metabolism and electrical activity, abnormalities of intracellular Ca2 + channels contribute to anoxia brain damage after the onset of some epilepsy or cerebral ischemia. Can be predicted. Cell loss is
It is said to be a characteristic property of degenerative brain disorders including Alzheimer's disease. It is well established that neuronal cell death is secondary to abnormally elevated cytosolic Ca2 +, especially with excitatory glutamate receptor activation (eg in epilepsy). This strongly suggests that the release of stored Ca2 + contributes to neuronal injury and cell death in various environments. The protein of SEQ ID NO: 386 appears to be functionally associated with other SPRY-containing proteins such as the ryanodine receptor, malenostrin / pyrin, SplA, midline-1 / FXY, and butyrophilin. Therefore, the protein is derived from intracellular Ca2 + storage organelles such as the endoplasmic reticulum and, in muscle, the sarcoplasmic reticulum (SR).
In addition to being associated with Ca2 + release, it appears to be involved in microtubule binding.
Preferred polypeptides of the present invention are fragments of SEQ ID NO: 386 having any of the biological activities described herein.

In one embodiment, the proteins and nucleic acids can be used to specifically detect fetal brain and uterine cells, as the proteins are expressed in fetal brain and uterus. For example, the proteins of the invention or portions thereof can be used to synthesize specific antibodies using techniques known to those of skill in the art. Such tissue-specific antibodies can then be used to identify tissues of unknown origin, such as forensic samples or tumor tissues that have metastasized and differentiated into different body sites, etc. Can be used to distinguish between tissue types. In addition, the proteins can be used to specifically label microtubules in cells. In other embodiments, the proteins of the invention or portions thereof can be used to regulate intracellular Ca2 + levels. Changes in intracellular Ca2 + homeostasis have significant effects on multiple cellular functions including secretion, contraction-relaxation, motility, metabolism, protein synthesis, modification and folding, gene expression, cell cycle progression and apoptosis. Intracellular
The ability to modulate Ca2 + levels provides a means to alter either of these cellular functions in vitro or in vivo. Such an ability can be used in numerous applications, for example, to manipulate the behavior of cultured cells in vitro (eg, growth rate, secretion, survival, etc.) as well as to treat multiple diseases associated with altered Ca2 + signaling in vivo. Has broad utility in prevention, prevention, or diagnosis. The activity or expression level of the protein of the present invention can be modulated by any of a number of methods, and examples thereof include the protein itself, nucleic acid encoding the protein, antibody, antisense sequence, dominant negative protein, and protein. A compound or the like that suppresses the activity or expression of can be administered to cells or patients and modulated. The effect of such formulations on intracellular intracellular calcium flux has been demonstrated by the use of tracers, light scatter and fluorescence quenching, and channel reconstitution into planar bilayers, for endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR). ) Can be detected using standard methods, which also include studying the permeation of calcium release through channels. In addition, targeted recombinant photoproteins allow direct measurement of Ca2 + in organelles (Montero
1995; EMBO J .; 14, 5467-5475). The invention further provides methods and compositions that use the proteins of the invention, or portions thereof, to diagnose, prevent, and / or treat some disorders in which the activity or recognition of the ryanodine receptor is diminished or excessive. Regarding things. These obstacles
Includes, but is not limited to, neurodegenerative diseases, cardiovascular disorders, myasthenia gravis, malignant hyperthermia, epilepsy, and central core disease. For example, in patients with myasthenia gravis, disease levels of anti-RyR antibodies are directly associated with severity (Skeie et al., 1993 E
ur. J. Neurol. 3; 136-140). Several lines of evidence suggest that RyR abnormalities are a major cause of many types of heart disease. In addition, the proteins of the invention can be used in the diagnosis of other diseases associated with SPRY protein dysfunction, such as familial Mediterranean fever and Opitz syndrome. Finally, SPRY-containing proteins have been implicated in embryonic development (eg, midline 1 protein) and the detection of mutations in the gene encoding SEQ ID NO: 386 or abnormal expression of the gene in the fetus is developmentally aberrant. Being an indicator of presence, the proteins and nucleic acids of the invention can be used to detect developmental disorders. For example, since the protein of SEQ ID NO: 386 is strongly expressed in fetal brain, it is likely that it plays a role in normal brain development in the uterus.

Furthermore, the present invention provides that the present protein is overexpressed as compared to a normal control tissue sample, resulting in neurodegenerative diseases, cardiovascular disorders, myasthenia gravis, malignant hyperthermia, epilepsy,
And a diagnostic assay for detecting altered levels of the protein of SEQ ID NO: 386 in various tissues as it may indicate the presence of multiple disease states such as Central Core disease. Assays used to detect levels of a polypeptide of the invention in a sample from a host are known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western Blot assays and Includes ELISA assay. <Proteins of SEQ ID NOs: 283 and 286 (internal designation numbers 174-38-1-0B6-CS_LA and 174-41-1-0-A6-CS LA)> SEQ ID NO: 283 encoded by the cDNA of SEQ ID NO: 42 The protein is overexpressed in the salivary gland and, to a lesser extent, in the bone marrow and shows homology to the immunoglobin (Ig) protein superfamily over the C-terminal length, which is eukaryotic (rabbit, rodent). (Including dental and human). In particular, the 468 amino acid long protein of the present invention, which is similar in size to the constant chain of an Ig-related protein, has 205 to 28
Positions 5 and 31-384 show two pfam-conserved immunoglobulin domains known to be involved in the basic structure of the light and heavy chains of the constant chain of immunoglobin. The Ig constant chain domain and single extracellular domain in each type of MHC chain is 100
It is known that they share homology of more than amino acids and are closely related (Orr HT,
Nature 282: 266-270 (1979)). All members of the Ig-related superfamily, including MHC class I alpha and beta-2 microglobulin, and MHC class II alpha and beta chains, have a prosite signature pattern (<FY> -xCx) conserved near the C-terminal cysteine. -<VA> -xH) is presented. This cysteine is involved in the disulfide bond between the light chain and the heavy chain, and is also found in the protein of the present invention (positions 380 to 386). Furthermore, the protein of the present invention displays the emotif Ig and major histocompatibility complex protein signatures at positions 319-336. Furthermore, the protein of the present invention comprises tapasin (GeneBank No. AF009510), T
It shows homology to chaperone-like proteins that are closely related to AP-binding proteins and is well conserved among eukaryotes, including chickens, rodents and humans.
Tapasin has been shown to increase the efficiency of antigen processing and presentation by mediating the association of MHC complex and TAP proteins with the endoplasmic reticulum and cell surface during the immune response (for a review, see Abele, R. . and Tampe, R., Bioch
et Biophysica Acta, 1999). Furthermore, the protein of the present invention is
Two transmembrane domains at positions 199-219 and 406-426 and a hydrophobic profile similar to the lyophobic range amino acids of human and mouse tapasin (Suling L., J. Biol. Chem., 274: 8649-8654, 1999) and a secretory signal peptide at positions 9-23. Both signatures are abundant in Ig-related proteins such as secretory antibodies or proteins that can present antigens. Furthermore, the invention also encompasses a variant of SEQ ID NO: 283 encoded by the cDNA of SEQ ID NO: 45 (SEQ ID NO: 286). The protein of SEQ ID NO: 286 is a 442 amino acid long protein with a C-terminal as short as 26 amino acids as compared to the protein of SEQ ID NO: 283. A variant of SEQ ID NO: 286 generated by a frameshift in the coding sequence (position 1445 of SEQ ID NO: 45) that reaches the stop codon in the corresponding protein is:
It exhibits the same features as described above for motifs, Ig signatures, functions, and potential uses.

The immunoglobulin (Ig) gene superfamily consists of the heavy and light chains of antibodies.
It contains numerous cell surface glycoproteins that share sequence homology with the V and C domains.
These molecules function as antigens, receptors for immunoglobulins and cytokines, and adhesion molecules, and play important roles in controlling the complex cellular interactions that occur in the immune system (AF Williams et al., Annu. Rev. Immuno. .6: 381-405, 19
88, T. Hunkapiller et al., Adv. Immunol. 44: 1-63, 1989; for a short report, see also Prosite entry PS00290). Introduction of the antigen into the host initiates a series of events culminating in the immune response. Furthermore, self-antigens may result in immune tolerance or activation of the immune response to self-antigens. The majority of immune responses are controlled by the presentation of antigens by major histocompatibility gene complex molecules. The MHC molecule binds to the peptide fragment derived from the antigen, forms a complex recognized by the T cell receptor on the surface of T cells, and forms MHC.
Causes the limiting T cell recognition phenomenon. Ability to react to a given antigen of the host (responsiveness)
Are influenced by a large variety of MHC molecules expressed by the host. Responsiveness is
Correlates with the ability of a specific peptide fragment to bind a particular MHC molecule. There are two types of MHC molecules, class I and class II, each containing two chains. In class I <2>, the alpha chain has three extracellular domains, one transmembrane region,
And one cytoplasmic tail. The beta chain (beta-2 microglobulin) consists of a single extracellular region. In class II <3>, both alpha and beta chains are 2
It consists of one extracellular domain, one transmembrane region, and one cytoplasmic tail. MHC
Class I molecules are expressed on the surface of whole cells and MHC class II molecules are expressed on the surface of antigen presenting cells. MHC class II molecules bind peptides derived from proteins formed extracellularly in antigen presenting cells. In contrast, MHC class I molecules bind peptides derived from proteins formed intracellularly. To present peptides in the environment of class II molecules, antigen presenting cells phagocytose the antigen in intracellular vesicles, where the antigen is cleaved and binds to MHC class II molecules and then to the surface of the antigen presenting cell. Will be returned. Major Histocompatibility Complex (MHC) Class I Molecules Have Antigenic Peptides on CD8 T
Present to cells (Townsend, A. et al., Nature: 340, 443-448)). Peptides are produced in the cytosol and then through the membrane of the endoplasmic reticulum, transport carriers associated with antigen processing (
TAP) is a trimeric complex consisting of TAP1, TAP2, and tapasin (TAP-A). TAP1 and TAP2 are required for peptide transport. Tapasin mediates the interaction of MHC class I HC beta-2 microglobulin with TAP, and this interaction is essential for peptide loading on MHC class I HC-beta-2 microglobulin (Suling et al. J. Biol. Chem., 274: 8649-8654). T cell receptor (TC
R) is a second antigen recognition molecule that recognizes the antigen bound to the MHC molecule. TCR
Recognition of MHC complexed with peptides by (MHC-peptide complex) can influence the activity of TCR-bearing T cells. Thus, the MHC-peptide complex is important in controlling T cell activity and thus the immune response.

Human cytomegalovirus (HCMV) is a beta-herpesvirus that causes a clinically significant illness in immunocompromised and immunosuppressed adults and in infants infected in utero or at birth (Alford). , CA, and WJ Bri
tt. 1990. Cytomegalovirus, p. 1981-2010. In DM Knipe and BN Fields
(Ed.), Virology, 2nd ed. Raven press, New York). In human cytomegalovirus (HCMV) -infected cells, the expression of the major histocompatibility complex (MHC) class I heavy chain of the cells is down-regulated. Here, down-regulation means MHC class I.
It is defined as a decrease in either heavy chain synthesis, stability or surface expression. Other DNs, including adenovirus, murine cytomegalovirus, and herpes simplex virus
A similar phenomenon has been reported in A virus (Anderson, M. et al., Cell 43: 2.
15-222, 1985; Burgert and Kvist, Cell 41: 987-997, 1985; Heise TM et al., J.
Exp. Med. 187: 1037-1046, 1998). In adenovirus and herpes simplex virus systems, even products of viral genes that are unnecessary for in vitro replication cause downregulation of MHC class I heavy chains. Sufficient (Anderson, M. et al., 1985, supra)
. The gene (s) involved in the downregulation of class I heavy chains by mouse cytomegalovirus has not yet been identified. The proteins of SEQ ID NOs: 283 and 286 are members of the immunoglobulin superfamily and are thereby involved in immune responses, cellular proteolysis, cell proliferation and differentiation, pathogen recognition, apoptosis, and other processes associated with the Ig superfamily. , Seems to play some role. Furthermore, the protein of the present invention, as a new chaperonin-like protein that binds to MHC I and TAP proteins, is closely associated with antigen processing and presentation systems in the context of peptide assembly and translocation of foreign peptides through the endoplasmic reticulum and cell surface membranes. It is thought to be related to. Low homology with the TAP protein family (30%) is due to Sul
As described by ing et al., supra, it is believed to represent the specificity of the interaction of the proteins of the invention with MHC proteins and / or TAP-related proteins. Preferred polypeptides of the invention are 9-23, 199-219, 205-285 of SEQ ID NO: 283.
, 318-384, 319-336, 380-386 and 406-426. Other preferred polypeptides of the present invention are fragments of SEQ ID NO: 283 having any of the biological activities described herein.

In one embodiment, the present invention provides a protein of the present invention or a part thereof as a marker protein for selectively identifying tissues such as salivary glands and bone marrow that strongly express the protein of the present invention. It relates to methods and compositions to be used. For example, the proteins of the invention, or portions thereof, can be used to synthesize specific antibodies using techniques known to those of skill in the art, including those described herein. Such tissue-specific antibodies can then identify tissues of unknown origin, such as differentiated tumor tissues that have metastasized to forensic samples, different body sites, etc., or use immunochemistry to distinguish different tissue types in tissue sections. Can be used for In another embodiment, the present invention provides a method of using the protein of the present invention for visualizing intracellularly proteins and peptides involved in the antigen recognition system by their physical interaction with the protein of the present invention. Regarding For example, the protein can be used to detect the presence and / or localization of MHC peptides and TAP-like proteins in cells. The protein of the invention, and thus any protein with which it can interact, binds to a specific antibody and contains a protein of the invention and an easily detectable moiety such as an epitope tag, biotin, or green fluorescent protein. Labeling can be done using any of several methods, including those that form fusion proteins. In other embodiments, the polynucleotide or polypeptide sequences of the invention, or portions thereof, can be used in the diagnosis of disorders associated with loss of regulation of expression of the proteins of the invention, preferably of the MHC protein system. Includes, but is not limited to, use in diagnosing deficiency. Examples of such disorders include acquired immunodeficiency syndrome (AIDS), X-linked Burton's agammaglobinemia, and unclassifiable immunodeficiency disease (CVI).
, DiGeorge syndrome (hypothyroidism), thymic dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease (SCID), immunodeficiency with thrombocytopenia and eczema (Wiscott-Aldrich syndrome), Cheejack・ Higashi syndrome, chronic granulomatosis, hereditary angioneurotic edema, immunodeficiency associated with Cushing's disease, Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis Disease, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, incidental lymphopenia with lymphotoxin , Fetal erythroblastosis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia , Irritable bowel syndrome, multiple sclerosis, myasthenia gravis,
Myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura Disease, ulcerative colitis, uveitis, Werner syndrome, cancer complications, hemodialysis and extracorporeal circulation, leukemias such as multiple myeloma, and lymphomas such as Hodgkin's disease; arteriosclerosis, atherosclerosis , Bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD
), Myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, cell proliferative disorders such as primary thrombocythemia, and adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma Cancers including, among others, adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate, salivary gland, Cancers of the skin, spleen, testis, thymus, thyroid, and uterus; and adenovirus, arenavirus, buniavirus, calicivirus, coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvo Virus, papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus,
Infection with rhabdoviruses and viral agents classified as togaviruses; Streptococcus pneumoniae, Staphylococcus, Streptococcus, Bacillus, Corynebacterium, Clostridium, Neisseria meningitidis, Neisseria, Listeria, Moraxella, Kingella, Haemophilus , Legionella, Bordetella, Shigella, Salmonella, and Gram-negative enterobacteria including Campylobacter, Pseudomonas, Vibrio, Brucella, Francisella, Yersinia, Bartonella, Nocardia, Actinomycetes, Actinomycetes, Helical bacterium, Infection with bacterial factors categorized into rickettsia, chlamydia, and mycoplasmas; Aspergillus, Blastomyces, Dermatophytes, Cryptococcus, Coccidioideus, Malassezia, Histoplasmas, and other fungal factors that cause a variety of mycosis. Infections with fungal agents to classify; and parasites Entamoeba, Leishmania, Trypanosoma, Toxoplasma, Pneumocystis carinii, Giardia, which cause varieties or malaria, Trichomonas, Trichinella Infections such as infections by parasites that are classified into tissue nematodes such as tissue nematodes, intestinal nematodes such as roundworm, lymphatic filarial nematodes, schistosomes, and tapeworms. , But not limited to these.
To investigate aberrant expression of the protein associated with any of these disorders,
The levels of the subject polynucleotides or polypeptides can be assayed using standard methods including Southern or Northern analyses, dot blots or other membrane-based techniques, PCR methods, dipsticks, pins and ELISA assays. Alternatively, it can be detected in cells and in microarrays. Any of these methods may be used to diagnose a disorder characterized by altered expression of the protein of SEQ ID NO: 283 or 286, such as the disorders described above, for the protein of SEQ ID NO: 283 or 286, or SEQ ID NO: 28
It can be used as an assay to monitor patients being treated with agonists, antagonists or inhibitors of 3 or 286 proteins. Antibodies useful for diagnostic purposes can be prepared, for example, as described in US Pat. No. 6,135,941. Diagnostic assays for the protein of SEQ ID NO: 283 or 286 include methods of detecting the protein of SEQ ID NO: 283 or 286 in human body fluids or extracts of cells or tissues using antibodies and labels. Antibodies can be used with or without modification and can be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of reporter molecules are known in the art and can be used, some of which are described above.

In another embodiment, a protein of SEQ ID NO: 283 or 286 or a fragment or derivative thereof is administered to a subject to diagnose, treat or prevent an immune disorder associated with decreased expression or activity of a protein of the invention. it can. Examples of such disorders are acquired immunodeficiency syndrome (AIDS), X-linked Burton's agammaglobinemia, nonclassifiable immunodeficiency (CVI), DiGeorge syndrome (thymic dysgenesis), thymus Dysplasia, isolated IgA deficiency, severe combined immunodeficiency (SCID), immunodeficiency with thrombocytopenia and eczema (Wiscott-Aldrich syndrome), Chejack-Higashi syndrome, chronic granulomatosis, hereditary blood vessels Neuroedema, immunodeficiency associated with Cushing's disease, Addison's disease, adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune Thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, accidental lymphopenia with lymphoid toxin, fetus Erythroblastosis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis , Myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenia Purpura, ulcerative colitis, uveitis, Werner syndrome, cancer complications, hemodialysis and extracorporeal circulation, leukemias such as multiple myeloma, and lymphomas such as Hodgkin's disease Not limited. In addition, disorders such as those described above that are associated with decreased protein expression or activity can be treated by administering to a patient a polynucleotide sequence encoding a protein of the invention, eg, inserted into a suitable vector. . In other embodiments, compounds that increase the activity of the proteins of the invention or their expression can be administered to a patient to treat or prevent any of the above disorders. In a further embodiment, a protein of the present invention for treating or preventing an immune disorder associated with increased expression or activity of the proteins of SEQ ID NOs: 283 and 286, including but not limited to autoimmune disease or graft rejection. Can be administered to the subject. In one embodiment, an antibody that specifically binds to a protein of the invention is directly or as an antagonist, or indirectly to cells or tissues that express the protein of the invention, such as salivary gland tissue or bone marrow tissue. It can be used as a targeting or delivery mechanism to move a dispensed substance. In addition, specific protein inhibitors such as sense, antisense nucleotides, GSEs, ribozymes, antibodies or small compounds may be administered to suppress the expression of the proteins of the invention.

[0386] In another embodiment, SEQ ID NO: 283 for the treatment or prevention of cell proliferative disorders.
Can be administered to a subject to be treated. Such disorders include arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (
MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and In particular, adrenal gland, bladder, bone, bone marrow, brain, breast, neck, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate, salivary gland, skin, spleen , But not limited to, cancer of the testis, thymus, thyroid and uterus. In one embodiment, an antibody that specifically binds to a protein of the invention is used to transfer the dispensed drug to a cell or tissue that expresses the protein of the invention, either directly as an antagonist or indirectly. It can be used as a targeting or delivery mechanism. In another example, sense, antisense nucleotides, GSE, or ribozyme designed from the nucleotides of the present invention may be administered to suppress the expression of the proteins of the present invention. <Protein of SEQ ID NO: 411 (Internal Designation Number 181-10-1-0-C9-CS)> The protein of SEQ ID NO: 411 encoded by the cDNA of SEQ ID NO: 170 is highly expressed in fetal liver. The protein of the invention is homologous to the benzodiazepine receptor / isoquinoline binding protein (PBR / IBP) from human, bovine and mouse (Gen).
Bank deposit numbers are M36035, M64520 and L17306, respectively. SEQ ID NO: 411 of 17
The 0 amino acid protein is similar in size and hydropathy to the known peripheral PBR / IBP benzodiazepine receptor / isoquinoline binding protein. Similar to the known peripheral PBR / IBP benzodiazepine receptor / isoquinoline binding protein, the protein of the present invention binds at positions 3-23, 45-65, 82-102, 105-125 and 130-150.
It has five latent transmembrane domains. Furthermore, the protein of the present invention has a recently identified putative cholesterol recognition / interaction amino acid common pattern (-L / V- (X)
In the range of 11 amino acids corresponding to (1-5) -Y- (X) (1-5) -R / K-) (starting from V144 to R154
End, <End etrinology 1998 Dec; 139 (12): 4991-
See 7>. Peripheral benzodiazepine receptor (PBR) is an 18-kDa protein containing the binding site for benzodiazepines, which is distinct from GABA neurotransmitter receptors <Papadop
oulos, V. (1993) Endocr. Rev. 14: 222-240>. Expression of PBR was found in all tissues tested. However, it is the most abundant in steroidogenic cells and is also found primarily on the outer mitochondrial membrane <Anholt
, R et al. (1986) J. Biol. Chem. 261: 576-583>. PBR is thought to associate with a multimeric complex composed of an 18-kDa isoquinoline-binding protein and a 34-kDa pore-forming voltage-gated anion channel protein that is selectively located at the mitochondrial outer / inner membrane contact site <McEnery, MW et al., Proc. Natl. Acad.
Sci. USA. 89: 3170-3174; Garnier, M. et al., (1994) Mol. Pharmacol. 45: 201-.
211; Papadopoulos, V. et al. (1994) Mol. Cel. Endocr. 104: R5-R9>. Drug ligands of PBR, when bound to the receptor, simulate steroid synthesis in steroidogenic cells in vitro <Papadopoulos, V et al. (1990) J. Biol.
Chem. 265: 3772-3779; Barnea, ER et al. (1989) Mol. Cell. Endocr. 64:
155-159; Amsterdam, A. and Suh, BS (1991) Endocrinology 128: 503-51
0>. Similarly, in vivo studies have shown that high-affinity PBR increases plasma steroid levels in hypophysectomized rats <Amri, H. et al. (1996) Endocrinology.
137: 5707-5718>. Further in vitro studies in isolated mitochondria have shown that PBR ligands, drug ligands, or endogenous PBR ligands (polypeptide diazepam binding inhibitors (DBI) <Papadopoulos, V. et al. (1997) Steroids 62: 2).
1-28>) provided evidence that it stimulates pregnenolone formation by increasing the rate of cholesterol translocation from the outer mitochondrial membrane to the inner membrane <For a review, see Culty, M. et al., (1999) Journal of. Steroid Biochemistry and Molecu
See lar Biology 69: 123-130>.

A three-dimensional model was developed based on the amino acid sequence of 18-kDa PBR <Papadopoul
os, V. (1996): Leydig cells Payne, AH et al. (Editor) Cache River Pres
s, IL, pp 596-628>. This model has been shown to adapt to function as cholesterol molecules and channels, supporting a role for PBR in cholesterol transport. The role of PBR in steroidogenesis was also demonstrated by observing the inability of PBR-negative cells produced by homologous recombination to produce steroids <Papadopoulos, V. et al. (1997) J. Biol. Chem. .272: 32129-321
35>. Furthermore, cholesterol transport experiments in bacteria expressing the 18-kDa PBR protein provided confirmation of its function as a cholesterol channel / transporter <Papadopoulos, V. et al. (1997) supra>. In addition to its role in mediating cholesterol translocation across membranes, PBR is involved in cell growth and differentiation, chemotaxis, mitochondrial physiology, porphyrin and heme biosynthesis, immune response, anion transport and central nervous system GABAergic action. It is also involved in several other physiological functions, including sex regulation. <For a review, see Gavish, M. et al. (1999) Pharmaceutical Reviews 51: 629-650; Beurdeley-Thomas, A. et al.
(2000) Journal of Neuro-Oncology 46: 45-56>. Also, recent reports further indicate that PBR agonists are potent anti-apoptotic compounds. These findings suggest that this effect may represent a major function of this receptor (Bono, F. et al. (1999) Biochemical and Biophysical Research Comm.
unications 265: 457-461). PBR appears to be associated with stress and anxiety disorders. PBR is HPA axis,
It has been suggested to be involved in the control of several stress systems such as the sympathetic nervous system, the renin-angiotensin axis, and the neuroendocrine axis. In these systems, acute stress typically leads to an increase in PBR density, whereas chronic stimulation typically leads to a decrease in PBR density. Furthermore, in generalized anxiety disorder (GAD), fearful disorder (PD), generalized social phobia (GSP), and post-traumatic stress syndrome (PTSD), PBR density is typically in platelets. It is falling. In brains in which PBR associates with glial cells, neurodegenerative disorders, and neurotoxicity and traumatic-ischemic brain injury, have increased PBR in specific brain areas. <For a review, see Gavish, M. et al. , (1999) supra>. A reduction in peripheral benzodiazepine receptors at postmortem autopsy in patients with chronic schizophrenia has also been reported in the literature, suggesting that reduced PBR density in the brain may be involved in the pathophysiology of schizophrenia. To do. Increased PBR levels in autopsy brain tissue from PSE patients (portal circulation encephalopathy patients) have been reported, thus activation of PBR is a characteristic occurrence in patients with portal circulation encephalopathy (PSE) in the central nervous system. Supporting the Theory of Contributing to Mechanisms <Kurumaji, A. et al. (1997) J. Neural Transm 104: 1361-1370; Butt
erworth RF (2000) Neurochemistry International 36: 411-416.

In addition to its involvement in neuropathy discussed above, PBR is also involved in the control of tumor cell growth <for a review, Gavish, M. et al. (1999) supra; Beurdeley. -
Thomas, A. et al. (2000) supra; Hardwick, M. (1999) Cancer Research 59: 831.
-842; Venturini, I. et al. (1998) Life Sci. 63: 1269-80; Carmel I et al. (19
99) Biochem Pharmacol 58: 273-8>. The invasive and metastatic potential of human breast tumor cells is proportional to the level of PBR expressed. Further, it has been proposed that PBR should be used as a tool / detection marker for the detection, diagnosis, prognosis and treatment of cancer <WO 99/49316, the entire text of which is incorporated herein by reference. I'm doing>. A number of ligands have been described to bind to peripheral benzodiacepine receptors with varying affinities. Some benzodiazepines, such as Ro 5-4864 <4-chlorodiazepam>, diazepam and structural analogs, are potent as well as selective PBR ligands. Exogenous ligands also include 2-phenylquinolinecarboxamide (PK11195 series), imidazo <1,2-a> pyridine-3-acetamide (Alpidem series) and pyridazine derivatives. Some endogenous compounds, including porphyrins and diazepam binding inhibitors (DBI), bind PBR with nanomolar (nM) and micromolar (μM) affinities <for a review, see Gavish, M
., Et al., (1999) supra; Beurdeley- Thomas, A. et al., (2000) supra>. The protein of SEQ ID NO: 411 is a new peripheral benzodiazepine receptor. Therefore, it plays a channel function that mediates the movement of cholesterol across the membrane,
It is involved in steroidogenesis, cell growth and differentiation, chemotaxis, mitochondrial physiology, protection against apoptosis, porphyrin and heme biosynthesis, immune response, anion transport and GABAergic regulation of the central nervous system. In one embodiment, a preferred polypeptide of the invention comprises the amino acids of SEQ ID NO: 411 from positions 144-154. In another embodiment, the invention provides a polypeptide comprising the sequence of SEQ ID NO: 411. Other preferred polypeptides of the present invention include the bioactive fragments of SEQ ID NO: 411. A biologically active fragment of the protein of SEQ ID NO: 411 has any of the biological activities described herein associated with PBR. In another embodiment, the polypeptide of the invention is encoded by clone 181-10-1-0-C9-CS.

One embodiment of the invention uses the protein of the invention, or a bioactive fragment thereof, for the development, identification and / or selection of agents capable of modulating the expression or activity of the protein of the invention, Providing compositions and methods. Agents that modulate PBR / IBP activity of the present invention include, but are not limited to, antisense oligonucleotides, ribozymes, drugs, and antibodies. These agents can be made and used according to methods known in the art. The proteins of the invention, or biologically active fragments thereof, can also be used in screening assays for therapeutic compounds. Various drug screening techniques can be used. In this aspect of the invention, the protein or bioactive fragment thereof is free in solution, attached to a solid support, recombinantly expressed on the cell surface, or chemically attached to the cell surface. Alternatively, it may be localized in the cell. The formation of a binding complex between the protein of the invention or a biologically active fragment thereof and the compound to be tested can then be determined. In one embodiment, the method utilizes a eukaryotic or prokaryotic host cell stably transformed with a recombinant nucleic acid expressing a PBR / IBP polypeptide or bioactive fragment thereof. The transformed cells may be live or fixed. Candidate drugs or compounds for modulating PBR / IBP, or biologically active fragments thereof, are screened against such transformed cells in binding assays known to those of skill in the art. Alternatively, assays such as those taught by International Patent Application Publication WO 84/03564 by Geysen HN, published September 13, 1984, the entire text of which is incorporated herein by reference, may be used for PBR / IBP,
Alternatively, it can be used to screen for peptide compounds that demonstrate binding affinity for, or ability to modulate, biologically active fragments thereof. In another embodiment, competitive drug screening assays using neutralizing antibodies are of the invention.
For binding to PBR / IBP protein, or a biologically active fragment thereof,
It specifically competes with the test compound. Other embodiments of the invention provide compositions and methods that selectively modulate the expression or activity of the proteins of the invention. Modulation of PBR / IBP treats and / or treats PBR or PBR / IBP-related diseases or biochemical abnormalities.
Or allow for successful management. Antagonists capable of decreasing or suppressing the expression or activity of the protein of the present invention are useful for treating diseases associated with elevated PBR / IBP levels, increased cell proliferation, or increased cholesterol transport. Therefore,
The present invention provides methods of treating various diseases or disorders including, but not limited to, cancer, especially liver cancer and patients with portal circulatory encephalopathy.

Alternatively, the invention provides a method of treating a disease or disorder associated with reduced PBR / IBP protein levels. Therefore, the present invention provides schizophrenia, chronic stress, GA
Provide a method of treatment of diseases including but not limited to D, PD, GSP and PTSD. Other diseases treatable by the PBR / IBP agonists of the present invention include diseases associated with decreased cell proliferation such as delayed development. Furthermore, since the PBR / IBP of the present invention can also transport cholesterol into cells, the present invention can also be used to increase cholesterol transport into cells.
Diseases associated with cholesterol transport deficiency require lipoid adrenal hyperplasia and increased production of cholesterol-requiring compounds such as myelin and myelination, such as Alzheimer's disease, spinal cord injury, and cerebral neuropathy Including diseases <Snipes, G. and Suter, U. (1997) Cholesterol and myelin Robert Bittman
Editing, subcellular biochemistry vol. 28, pp. 173-204, Plenum Press, New York>. P
A method of treating disorders associated with reduced BR / IBP levels can be practiced by introducing an agonist that stimulates the expression or activity of the proteins of the invention. In one embodiment, the method for raising PBR / IBP levels in a tissue or cell type comprises introducing a bioactive polypeptide into a target cell type, a nucleic acid encoding a protein of the invention, or an organism thereof. This can be done by using the active fragment. Vectors useful in such methods are known to those of skill in the art, as are methods for introducing such nucleic acids into target tissues. Agents that stimulate or inhibit the activity of the proteins of the invention include, but are not limited to, agonist and antagonist drugs, respectively. These drugs can be obtained using any of the various drug screening techniques discussed above. The antagonist of PBR / IBP encoded by SEQ ID NO: 170 is SEQ ID NO: 411.
Agents that reduce the level of expressed mRNA encoding the protein of These are RN
Ai, including, but not limited to, one or more ribozymes capable of digesting a protein of the invention, an mRNA, or an antisense oligonucleotide capable of hybridizing to an mRNA encoding PBR / IBP of SEQ ID NO: 411. It is not done. The antisense oligonucleotide is used as DNA trapped in a proteoliposome containing a viral envelope receptor protein <Kanoda, Y. et al. (1
989) Science 243: 375> Alternatively, it can be administered as part of a vector that can be expressed in target cells to provide antisense DNA or RNA. Vectors that are expressed in specific cell types are known in the art. Alternatively, the DNA may be injected with the carrier. The carrier can be a protein such as a cytokine, eg, interleukin 2 or polylysine glycoprotein carrier. Methods of making and using carrier proteins, vectors, and polylysine carrier systems are known in the art.
Alternatively, nucleic acids encoding antisense molecules can be coated on gold beads and introduced into the skin, eg, by gene gun <Ulmer, JB et al. (1993) Science 259:
1745>.

Antibodies or other polypeptides capable of reducing or suppressing PBR / IBP activity can be provided in isolated and substantially purified form. Alternatively, antibodies or other polypeptides capable of suppressing or reducing PBR / IBP protein activity can also be recombinantly expressed in target cells to provide a modulating effect. In addition, compounds that suppress or reduce PBR / IBP protein activity of the present invention may be incorporated into biodegradable polymers that are implanted in the vicinity where drug delivery is desired. For example, a biodegradable polymer can be implanted at the tumor site or an antagonist / agonist containing biodegradable polymer can be implanted systemically to slowly release the compound. Biodegradable polymers and their uses are known to those of skill in the art (see, eg, Brem et al. (1991) J. Neurosurg. 74: 441-446). In other embodiments, the invention provides methods and compositions for detecting the expression level of mRNA of the proteins of the invention. Quantitation of mRNA levels of the PBR / IBP proteins of the invention can be useful in diagnosing or prognosing diseases associated with modified expression of the proteins of the invention. Assays for the detection and quantification of mRNA of the proteins of the invention are known to those skilled in the art (eg Maniatis, Fitsch and Sambrook, Molecular Cloning; Experimental Manual (1982), or Current Protocols in Molecular Biology, Ausubel, FM, et al. (Edit), Wiley & Sons, Inc). A polynucleotide probe or primer for detecting mRNA of the protein of SEQ ID NO: 411 can be designed from the cDNA of SEQ ID NO: 170. Methods for designing probes and primers are known in the art. In another embodiment, the invention provides a diagnostic kit for the detection of mRNA of a protein of the invention in cells. The kit comprises one or more containers consisting of oligonucleotide primers for detecting the protein of the invention in a PCR assay or one or more polynucleotide probes for detecting mRNA of the protein of the invention by in situ hybridization or Northern analysis. Including a package with a container. The kit optionally includes a container of various reagents used in various hybridization assays. The kit also optionally includes one or more of the following items: polymerizing enzymes, buffers, instructions, controls, or detection labels. The kit also optionally contains a container consisting of reagents mixed in suitable proportions for performing the hybridization assay according to the invention. The reagent container preferably contains the reagents in a unit amount which omits the measuring step when carrying out the method.

In another embodiment, the present invention relates to methods and compositions for detecting and quantifying the levels of proteins of the present invention present in a particular biological sample. These methods are useful for diagnosis or prognosis of diseases in which the expression level of the protein of the present invention is modified. Diagnostic assays for detecting the proteins of the invention may include biopsies, in situ assays of cells from organ or tissue sections, or aspirates of cells from tumor or normal tissue. In addition, the assay can be performed on cell extracts from organs, tissues, cells, urine, or semen or blood or other body fluids or extracts. The assay for quantifying PBR / IBP of SEQ ID NO: 411 can be performed according to a method known in the art. Typically, these assays involve contacting the sample with a ligand of the invention, or an antibody (polyclonal or monoclonal) that recognizes the protein of the invention or a fragment thereof, and the protein of the invention and ligand present in the sample. Alternatively, it includes detecting the complex formed between the antibodies. Ligand and antibody fragments can also be used in binding assays under conditions where these fragments can specifically interact with the BRP / IRPs of the invention. Further, the BRP / IR of the present invention
Antibodies that bind P can be labeled according to methods known in the art. Labels useful in the present invention include, but are not limited to, enzyme labels, radioisotope labels, paramagnetic labels, and chemiluminescent labels. A typical technique is Kenn
edy, JH, et al. (1976) Clin. Chim. Acta 70: 1-31; and Schurs, AH.
(1977) Clin. Chim. Acta 81: 1-40. The invention also provides methods and compositions for the identification of metastatic tumor masses. In this aspect of the invention, polypeptides and antibodies that bind the polypeptides of the invention can be used as markers for identifying metastatic tumor masses. Primary liver metastatic tumors overexpress the PBR / IBP of SEQ ID NO: 411, but neoplastic tumors or tumors originating in other tissues are not expected to carry the PBR / IBP of SEQ ID NO: 411. <Protein of SEQ ID NO: 397 (Internal designation number 160-28-4-0-C4-CS)> SEQ ID NO: 397 encoded by the cDNA of SEQ ID NO: 156 (clone 160-28-4-0-C4-CS) Protein exhibits homology to the ADP ribosylation factor (ARF) family of proteins. The ARF family includes ADP ribosylation factor (ARF) and ARF
The ARF family of proteins is a member of the Ras superfamily. Proteins belonging to the Ras superfamily have a molecular weight of 18-30 kDa and function in various cellular processes including, but not limited to, signal transduction, growth, immunity, and protein trafficking.

ARF is a monomeric GTP-binding protein and is structurally related to both the G protein alpha subunit and the Ras protein. ARF family members are
It shares more than 60% sequence identity, appears ubiquitous in eukaryotes, and is highly evolutionarily conserved throughout eukaryotes. Immunologically, they are localized in the Golgi apparatus of several cell types (Stearns et al., Proc. Natl. Acad. Sci.
A) 87: 1238-1242 (1990)). As an allosteric activator, the ARF protein enhances the ADP-ribosyltransferase activity of cholera toxin (Noda et al., Biochim.
Biophys. Acta 1034: 195-199 (1990)). ARF has also been shown to act as a regulatory molecule or a "switch" that links two processes, such as the process of vesicle division from the donor compartment and fusion with the receptor compartment (Rothman, J.
E. and Wieland, FT Science 272: 227-234 (1996)). ARF family members are divided into three classes, classes I-III, according to their size and sequence homology. Class I includes ARF1, ARF2, and ARF3; Class II includes ARF4 and ARF5; and Class III includes ARF6. These classes occupy different subcellular locations and are involved in different transport pathways. Class I ARF is localized in the Golgi and is involved in the regulation of ER-Golgi and intra-Golgi transport. Class I ARF is also involved in the recruitment of cytosolic coat proteins to the Golgi cell membrane during transport vesicle formation. Class III (eg, ARF6) is localized to tubular vesicle compartments, secretory granules, and plasma membranes, where it is involved in the regulation of secretion and recycling. Class II ARFs are thought to be cytosolic, but their role has not been elucidated. (Radhakrishna, H. and Donaldson, JGJ C
ell Biol. 139: 49-61 (1997)). AFR function is generally controlled by the GDP-GTP cycle. For example, ARF1 is cytosolic in the GDP-bound state, but associates with the membrane in the GTP-bound state. Guanine nucleotide exchange factor (GEF) in the donor compartment is ARF1
To the membrane. At the membrane, GTP-ARF1 recruits coat proteins and puts them together to construct a globular capsule, during which process vesicles bud. After germination, ARF1 is dissociated from the membrane by hydrolysis of bound GTP. Dissociation of AFR1 renders the coating unstable and results in similar dissociation. (Rothman, supra.) Members of the ARF multigene family display different phospholipid and detergent requirements when expressed as recombinant proteins in E. coli (Price et al., J. Biol.
Chem. 267: 17766-17772 (1992)). Certain lipids and / or detergents such as SDS, cardiolipin, dimyristoylphosphatidylcholine (DMPC) / cholate enhance ARF activity (Bobak et al., Biochemistry 29: 855-861 (1990); N).
oda et al., Biochim. Biophys. Acta 1034: 195-199 (1990); Tsai et al., J. Biol.
Chem. 263: 1768-1772 (1988)). ARF also activates phospholipase D (PLD), a membrane-bound enzyme involved as an effector of some growth factors.
PLD1 has been shown to be activated by a variety of G protein regulators such as PKC (protein kinase C) and ADP-ribosylating factor (ARF). PK
C and ARF can regulate G proteins alone or together in a synergistic manner. Recently, the role of ARF in microtubule formation has also been demonstrated. ADP-ribosylation of tubulin almost completely blocks self-assembly of this protein in the brain (Terashima M. et al; J. Nutr Sci Vitaminol 45: 393-400 (1999).
).

In general, the differences in the diverse ARF sequences are concentrated in the amino terminal region and the carboxyl portion of the protein. Only three of the 17 amino acids at the amino terminus have been shown to be identical between ARFs, and four amino acids in this region of ARF1-5 are absent in ARF6 (Tsuchiya et al., J. . Biol. Chem. 26
6: 2772-2777 (1991)). It has been reported that the amino-terminal region of the ARF protein forms an alpha helix and this domain is required for membrane targeting, lipid interaction and ARF activity ((Kahn et al., J. Biol. Chem. 267. : 1303
9-13046 (1992)). Schliefer et al. (J. Biol. Chem. 257: 20-23 (1991)) describe apparently larger proteins with ARF-like activity. ARF-like protein,
Alternatively, ARL has been found in different species. Some ARLs lack ADP-ribosyltransferase-enhancing activity, and ARLs may differ in GTP-binding requirement and GTP-hydrolase activity when compared to various ARF isoforms .
For example, the mammalian ARL, ARP, is 33-39% identical to ARF family members, but ARPs by virtue of its ability to hydrolyze bound GTP in the absence of other proteins. Different from ARP proteins, unlike ARF, typically associate with the plasma membrane instead of the cytosol (Schurmann, AJ
Biol. Chem. 270, 30657-30663 (1995)). ARF family members have been implicated in several disease processes such as Lowe syndrome, congenital cataracts, renal tubular dysfunction and X-linked disorders characterized by neuronal deficits. These disorders are likely due to the inability of ARF to recruit to the Golgi membrane (Suchy, SF et al., Hum. Mol. Genet. 4: 2245-2250 (1995), Londono I.
Et al., Kidney Int. 55: 1407-1416 (1999)). Regulation of ARF has also been implicated in cystic fibrosis, Dent's disease, diabetes, and autosomal dominant polycystic kidney disease (Marshansky, V. et al., Electrophoresis 18: 2661-2676 (1997)). . In one embodiment, the novel human ARF-related protein of SEQ ID NO: 397, encoded by clone 160-28-4-0-C4-CS, and related polynucleotides are secreted, exocytotic, endocytotic and other Provide new compositions useful in the diagnosis, treatment and prevention of "selective disorders".

The present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 397 or clone 160-28-4-0-C4-CS, or a biologically active fragment thereof. The target intact protein is 173 amino acids long, has an ARF family amino acid motif (Pfam), and further has an ATP / GTP binding site motif, AP loop (PS00017). The protein of SEQ ID NO: 397 or clone 160-28-4-0-C4-CS was also labeled with human ARL1 (P4
0616), ARD-1 (R66033) and ARF6 (GI 178989) with chemical and structural similarities (31%, 31% and 27% identity, respectively). The amino acid length of SEQ ID NO: 397 is similar to that of ARF described above. The bioactive fragment of SEQ ID NO: 397 has one or more of the bioactivity typically associated with a full length protein. In one embodiment, the protein is encoded by clone 160-28-4-0-C4-CS. The present invention also provides a variant of the protein of SEQ ID NO: 397 or clone 160-28-4-0-C4-CS. The variant comprises at least about 80%, preferably at least about 90%, and most preferably at least about 95% of the amino acid sequence of SEQ ID NO: 397 or clone 160-28-4-0-C4-CS. Have identity. The variant according to the invention has at least one functional and / or structural characteristic of ARF. The present invention also provides bioactive fragments of variant proteins. The present invention provides a polynucleotide encoding the protein of SEQ ID NO: 397 or clone 160-28-4-0-C4-CS, a variant of SEQ ID NO: 397 or clone 160-28-4-0-C4-CS, and a sequence. No. 397 or the bioactive fragment of clone 160-28-4-0-C4-CS and variants thereof. As will be apparent to those of skill in the art, a variety of different DNA sequences can encode the amino acid sequences of proteins, variants and bioactive fragments of said proteins and variants. Creating these alternative DNA sequences that encode proteins with identical, or substantially identical amino acid sequences,
It is within the skill of a person skilled in the art. These mutant DNA sequences are also within the scope of the invention. As used herein, a "substantially identical" sequence means a sequence having amino acid substitutions, deletions, additions or insertions that does not substantially affect biological activity. The present invention includes cytoskeletal, secretory, and inflammatory disorders / conditions comprising administering a composition comprising a therapeutically effective amount of the protein of SEQ ID NO: 397 or clone 160-28-4-0-C4-CS. Supply a method for treating. These methods are also described in SEQ ID NO: 39.
7 or a variant of clone 160-28-4-0-C4-CS, or SEQ ID NO: 397 or clone
Bioactive fragment of 160-28-4-0-C4-CS, or SEQ ID NO: 397 or clone 1
It can be carried out using a mutant of 60-28-4-0-C4-CS. Disorders / conditions treatable by the present invention include prostate cancer, brain and other tumors, Rho's syndrome, glomerulonephritis, chronic glomerulonephritis, renal tubular interstitial nephritis, hereditary X-linked renal primary diabetes insipidus, Autosomal dominant polycystic kidney disease (ADPKD), herpes gestation, herpes dermatitis, lupus erythematosus,
Crohn's disease, irritable bowel syndrome, and Addison's disease; cystic fibrosis, glucose-
Intravesicular cells, including secretory / endocytotic disorders such as galactose malabsorption syndrome, hypercholesterolemia, hyper and hypoglycemia, Gravis disease, goiter, and Cushing's disease; acquired immunodeficiency syndrome (AIDS) Conditions associated with transport abnormalities;
Allergies including hay fever, asthma, and urticaria (urticaria); autoimmune hemolytic anemia; multiple sclerosis; myasthenia gravis, rheumatoid and osteoarthritis; chesiac
East and Sjogren's syndrome; toxic shock syndrome; trauma tissue injury; virus,
Includes, but is not limited to, bacterial, fungal, parasite-like and protozoal infections.

In other embodiments, vectors capable of expressing the protein of SEQ ID NO: 397 or clone 160-28-4-0-C4-CS, or biologically active fragments thereof, including those described above, are provided. It can be administered to a subject to treat or prevent a disorder, including but not limited to: Alternatively, the vector can encode a variant, or bioactive fragment of a variant protein. SEQ ID NO: 397 or clone 160-28-4-0-C4-CS
, A variant, and / or a bioactive fragment of SEQ ID NO: 397 or clone 160-28-4-0-C4-CS, and / or a combination of variants can be administered to the subject. In a further embodiment, a pharmaceutical composition comprising a substantially purified protein of SEQ ID NO: 397 or clone 160-28-4-0-C4-CS (and / or a biologically active fragment thereof) is provided as a suitable pharmaceutical carrier. In combination with, it can be administered to a subject for the treatment or prevention of the above disorders. Alternatively, SEQ ID NO: 397 or clone 160-28-4-0-
A pharmaceutical composition comprising a substantially purified variant protein of C4-CS (and / or a biologically active fragment thereof) can be administered in the therapeutic regimens described above in combination with a suitable pharmaceutical carrier. As will be apparent to those of skill in the art, the protein encoded by SEQ ID NO: 397 or clone 160-28-4-0-C4-CS (and / or a biologically active fragment thereof) and SEQ ID NO: 397 or clone 160-28-. 4-0-C4-C
A therapeutically effective combination of variants of S (and / or biologically active fragments thereof) can be used in the therapeutic regimen described above in combination with a suitable pharmaceutical carrier. ARF is known to be involved in the controlled transport of vesicles. Thus, in another embodiment, the protein, variant, or SEQ ID NO: 397 or clone 160-28-4-0-C4-CS,
And / or bioactive fragments of said proteins and / or variants can be used as components of drug delivery vesicles such as colloids or liposomes. The protein, variant, and / or bioactive fragment of said protein and / or variant of SEQ ID NO: 397 or clone 160-28-4-0-C4-CS is incorporated into the lipid membrane of liposomes for specific targeting. Can work as a drug. Methods of such drug delivery system design are known to those of skill in the art and can be performed according to conventional pharmaceutical principles (Smith HJ Drug Design and Introduction to Principles of Action (Intro
duction to the principles of drug design and action), 3rd edition. (1998):
Chien YW Novel Drug Delivery systems, Second Edition (1
992); Storm G. et al., J. Liposome Res. 4: 641-666 (1994); and Crommelin.
DJA et al., Adv. Drug Delivery Rev. 17: 49-60 (1995)).

In another embodiment of the invention, the polynucleotide encoding the protein of SEQ ID NO: 397 or clone 160-28-4-0-C4-CS can be used for therapeutic purposes. SEQ ID NO: 39
A polynucleotide encoding a protein fragment of 7 or clone 160-28-4-0-C4-CS can also be used in therapeutic treatment. In one embodiment, the complement of the polynucleotide encoding the protein of SEQ ID NO: 397 or clone 160-28-4-0-C4-CS can be used in situations where it is desirable to block the transcription of mRNA. Modification of gene expression is performed by complementing a control sequence, 5 ′, or regulatory region of a gene encoding a target protein or an antisense molecule (DNA, RNA).
Or PNA) can be obtained by designing. Such technologies are now well known in the art, and sense or antisense oligonucleotides or large fragments can be designed from various positions in the coding or control regions of the sequence encoding the protein of interest. Therapies using antisense technology are also known to those of skill in the art. Another embodiment of the invention provides a method of assessing PLD modulation using the ARF properties of a protein of interest. In another embodiment, an antibody that specifically binds the protein of SEQ ID NO: 397 or clone 160-28-4-0-C4-CS is diagnosed with a disorder characterized by expression of the protein or treated with the protein of interest. Can be used in an assay to monitor a patient. Methods for producing polyclonal and monoclonal antibodies are known in the art. Diagnostic assays that can be used in this aspect of the invention are ELIS
It is known in the art, including but not limited to A, RIA, and FACS. These assays also diagnose modified or aberrant expression levels of the polypeptide encoded by the human cDNA of SEQ ID NO: 397 or clone 160-28-4-0-C4-CS as compared to normal individuals. Or provide the basis for identification. These screening methods are also known to those skilled in the art. In other embodiments of the invention, the proteins of the invention, catalytic or immunogenic fragments thereof, or oligopeptides thereof can be used to screen compound libraries in various drug screening techniques. Fragments used in such screens may be free in solution, attached to a solid support, recombinantly expressed on the cell surface, chemically linked to the cell surface, or located between cells. May be. The formation of binding complexes between the protein of interest and the drug to be tested can be determined by methods known to those skilled in the art. Other techniques of drug screening provide high throughput screening of compounds with appropriate binding affinity for the protein of interest. (See, eg, Geysen et al. (1984) International Patent Application Publication WO 84/03564). In another embodiment of the invention, a polynucleotide encoding a protein of interest can be used for diagnostic purposes. The polynucleotides can be used to detect and quantify gene expression in biopsy tissues where expression of the protein of interest may correlate with disease or condition. Such diagnostic assays are well known in the art and can be used to monitor the regulation of the protein of interest during therapeutic intervention and / or to determine the presence or overexpression of the protein of interest. Examples of such conditions and disorders are provided above. The polynucleotide sequence encoding the protein of interest is prepared using, for example, Southern bodily fluid from a patient to detect expression modification of the protein of SEQ ID NO: 397 or clone 160-28-4-0-C4-CS. Or it can be used in Northern analysis, dot blots, or other membrane technologies; PCR technology; dipstick, pin, and ELISA assays; and microarrays. Such qualitative or quantitative methods are known in the art.

In a further embodiment, oligonucleotides or long fragments derived from the polynucleotide sequences described herein can be used as targets in microarrays. Microarrays can be used to simultaneously monitor expression levels of multiple genes and identify genetic variants, mutations, and polymorphisms. This information can be used to determine gene function, understand the genetic basis of the disorder, diagnose the disorder, and develop therapeutic agents and monitor their activity. Microarray
It can be prepared, used, and analyzed using methods known in the art (eg, Bre
nnan, TM et al. (1995) US Pat. No. 5,474,796; Heller, RA et al. (1997) Proc.
Natl. Acad. Sci. 94: 2150-2155; and Heller, MJ et al. (1997) US Pat. No. 5,605,662). Other embodiments of the invention provide nucleic acid sequences encoding a protein of interest that can be extended using partial nucleotide sequences and various PCR methods. This aspect of the invention provides a method for detecting upstream sequences such as promoters and control elements.
Methods of practicing this aspect of the invention are also known in the art. In other embodiments of the disclosed therapeutic regimens, the proteins, variants, bioactive fragments, antibodies, complementary sequences, or vectors of the invention can be administered in combination with other suitable therapeutic agents. Selection of appropriate agents for use in combination therapy is within the skill of those in the art. The combination of therapeutic agents can act synergistically to affect the treatment or prevention of the various disorders described above. In particular, purified protein is produced by antibodies,
Alternatively, it can be used to screen a drug library to identify those that specifically bind the protein of interest. Neutralizing antibodies are particularly preferred for therapeutic use. <Protein of SEQ ID NO: 287 (internal designation number 174-5-3-0-H7-CS)> Sequence encoded by a human cDNA which is SEQ ID NO: 46 (clone 174-5-3-0-H7-CS) The protein of number 287 is highly homologous to the human protein encoded by the CLN8 gene listed under Genbank accession number AF123757 (99%).
Amino acid identity above). The two proteins differ by two conserved amino acid substitutions: valine at position 155 is alanine and asparagine at position 225 is serine. In addition, the protein encoded by 174-5-3-0-H7-CS contains seven transmembrane domains. These domains are software TopPred I
Amino acids 25-45, 71-91, 100-120 and 133-1 as predicted by I
Located at 53 (Claros and von Heijne, CABIOS applic Notes, 10: 685-686 (19
94)). The protein encoded by SEQ ID NO: 287 also exhibits a signal peptide at positions 1-50 and a retention signal KKRP at positions 283-286.

CLN8 was recently identified by positional cloning (Ranta et al., Nat. Genet.
1999 Oct.; 23 (2): 233-6). CLN8 encodes a 286 amino acid putative transmembrane protein that has no homology to previously known proteins. A naturally occurring missense mutation at codon 24 (R24G at the border of the first putative transmembrane domain) resulted in EPMR
("Progressive Tencan with Mental Retardation", MIM 600143) is the molecular basis. EPMR, also known as Northern Epilepsy, is an autosomal characterized by normal early development, the onset of generalized tonic-clonic tencan seizures between ages 5 and 10, followed by progressive mental retardation. It is a recessive disorder. Neuropathological findings indicate that EPMR is a new member of the neuronal ceroid lipofuscinosis (NCL) group of neurodegenerative disorders. NCLs are a genetically heterogeneous group of progressive neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigment in diverse tissues. CLN8 is the eighth gene associated with the NCL group of neurodegenerative disorders. The homologous mouse gene (Cln8) was then sequenced (82% nucleotide identity with the human gene) to identify the region of the mouse genome associated with motor neuron degeneration, mouse mnd.
Localized to. Mnd is a naturally occurring mouse mutant with intracellular autofluorescent inclusion bodies similar to those observed in EPMR. A mutation was identified in mnd mouse DNA, which is a mouse ortholog of mnd CLN8 (Ranta et al. Nat Genet. 1999).
Oct; 23 (2): 233-6), mice containing a Cln8 mutation were shown to present a mouse model of NCL injury. Recent experimental evidence confirmed the transmembrane properties of the CLN8 protein (Lonka L et al., Hu
m Mol Genet 2000 Jul 1; 9 (11): 1691-7). CLN8 resides in the endoplasmic reticulum (ER) and its C
ER and ER via the KKXX ER recovery motif at the end (KKRP, amino acids 283-286)
-Recirculate the Golgi intermediate compartment (ERGIC). This motif is from sis Golgi to ER
It is recognized and bound by COPI, a vesicle-coating protein found in retrograde vesicles that delivers cargo to the. The 30 kD CLN8 protein is not processed during its maturation (particularly not N-glycosylated). EPMR-related R24G mutations do not modify cell localization in humans. The invention provides a polypeptide encoded by SEQ ID NO: 287 and bioactive fragments of said polypeptide. A composition comprising the polypeptide and a pharmaceutically acceptable carrier is also provided. Preferred polypeptides, and biologically active fragments thereof, have any of the biological activities or domains / motifs described herein, and / or include amino acids at positions 155-225, 283-286. In one embodiment, the protein / polypeptide of SEQ ID NO: 287 is encoded by clone 174-5-3-0-H7-CS.

ER / ERGIC cellular localization of the proteins of the invention can be used to target compounds to ER / ERGIC. This targeting is described by (Lonka L et al., Hum Mol Genet. 2000 Jul.
1; 9 (11): 1691-7), including any of the techniques known to those of skill in the art. In this aspect of the invention, the protein of SEQ ID NO: 287, or bioactive fragments thereof, can be used to target liposomes, vesicles, or colloids to the ER / ERGIC compartment to which the active agent is delivered. Methods of making and using targeted liposomes are known in the art. In other embodiments, liposomes containing the protein of SEQ ID NO: 287 may contain a second targeting agent for the specific selection of target cells. The second targeting agent is selected for its ability to specifically target cells or tissues. Thus, the second targeting agent may be specific for a tumor marker, such as HER2. Alternatively, a marker associated with a particular cell type may be used (eg CD34, CD4, CD8 etc.). In a preferred embodiment, the second targeting agent is an antibody. Active agents include, but are not limited to, chemotherapeutic agents, protein crosslinkers, protein synthesis inhibitors, antibacterial agents (eg, antibiotics), antiviral agents, and / or anthelmintic agents. The ability to combine COPI coaters is (
Cosson P, Letourneur F, Science. 1994 Mar 18; 263 (5153): 1629-31). In other embodiments, the invention provides, such as ER, ERGIC, and retrograde transport vesicles,
Methods and compositions for identifying specific cell compartments are provided. This embodiment provides an antibody that specifically binds the protein of SEQ ID NO: 287, or a biologically active fragment thereof, labeled with a detectable marker such as a gold particle, an enzyme, a radioisotope, or a paramagnetic label. ER, ERGIC, and retrograde transport vesicles can be identified in a sample according to known immunodiagnostic protocols. Antibodies, either monoclonal or polyclonal, can be produced according to known methods. In a preferred embodiment, the antibody binds the ER retention signal. In other embodiments, the proteins of the invention, or portions thereof, are for differential identification of tissue (s) or cell type (s) present in a biological sample, and asthma, pulmonary edema, atherosclerotic arteries. It can be used as a reagent for the diagnosis of diseases and conditions including, but not limited to, sclerosis, restenosis, occult stroke, thrombosis and hypertension. Similarly, the proteins of the invention, or biologically active fragments thereof, and antibodies thereto can provide immunological probes for differential identification of tissue (s) or cell type (s). Certain disorders or cell types (eg vascular tissue, cancerous and wound tissue) or body fluids (eg lymph, serum, plasma, urine, synovial fluid) in some of the disorders listed above, especially in the lungs and cardiovascular system And cerebrospinal fluid) or other tissue or cell samples taken from individuals with such disorders, compared to standard gene expression levels, ie, expression levels in normal tissues or body fluids from individuals without the disorder. Very high or low expression levels of the protein can be routinely detected.

Indeed, the first 80 amino acids of the protein of the invention are the two polypeptides claimed in WO 99/35158, the entire text of which is incorporated herein by reference. (SEQ ID NO: 98 and SEQ ID NO: 162, corresponding to Geneseq accession numbers Y38413 / Y38428 and Y38492, are overexpressed in lung and endothelial tissue). The distribution of this tissue in lung and endothelial tissues is characterized by the fact that the protein products described in WO 99/35158 are: asthma, pulmonary edema, pneumonia, atherosclerosis, restenosis, stroke,
It is shown to be useful in the treatment and diagnosis of cardiovascular and respiratory or lung disorders such as angina, thrombosis, hypertension, inflammation, and wound healing. Those conditions can be diagnosed by measuring the amount of the protein of the present invention in the sample. Therefore,
Antibodies raised against the protein of SEQ ID NO: 287, or immunogenic fragments of this protein can be used in diagnostic, prognostic, or screening assays, such as those taught in WO 99/35158. <The protein of SEQ ID NO: 270 (internal designation number 116-119-3-0-H5-CS)> The protein of SEQ ID NO: 270 encoded by the extended cDNA of SEQ ID NO: 29 is:
Human mitochondrial ATP synthase f subunit f or ATPK (EC 3.6.1.34)
(Swissprot accession number P56134) and is overexpressed in fetal kidney. The protein of SEQ ID NO: 270, which consists of 88 amino acid residues, is the software TopPred
Contains one transmembrane segment (positions 1-55) as predicted by II (Cla
Ros and von Heijne, CABIOS applic Notes, 10: 685-686 (1994)). The BLAST results show that amino acids 5 to 88 of the protein of the present invention and amino acid 10 of human ATP synthase f chain.
100% homology was found between ~ 93, indicating that exon 1 of the cDNA of SEQ ID NO: 29 is responsible for the difference between the two proteins (the latter three exons show 100% homology). ). Thus, the proteins of the present invention point to a new isoform of human mitochondrial ATP synthase subunit f. Interestingly, identical splice variants have been found in bovine, porcine and murine species. The mitochondrial electron transport (or respiratory) chain transports electrons from NADH to oxygen,
And a series of enzyme complexes in the mitochondrial membrane responsible for coupling this oxidation to ATP synthesis (oxidative phosphorylation). ATP then supplies the primary source of energy to drive the cell's multiple energy-requiring reactions. ATP synthase (F0 F1 ATP-degrading enzyme) is an enzyme complex at the end of this chain that interconverts the energy of an electrochemical hydrogen ion gradient across the mitochondrial membrane into either ATP synthesis or hydrolysis. , Acts as a reversible conjugate device. This gradient is generated by other enzymes in the respiratory chain in the process of electron transfer from NADH to oxygen. When the cell's energy requirements are high, electron transfer from NADH to oxygen produces an electrochemical gradient across the mitochondrial membrane. Transfer of hydrogen ions from the outside to the inside of the membrane drives ATP synthesis. Under low energy requirements and in the presence of excess ATP, this electrochemical gradient is reversed and ATP synthase hydrolyzes ATP. Hydrolysis energy is used to pump hydrogen ions out of the mitochondrial matrix. ATP synthase is thus a double complex consisting of the F0 moiety, which is a transmembrane hydrogen ion carrier or pump, and the F1 moiety, which catalytically synthesizes or hydrolyzes ATP. The mammalian ATP synthase complex consists of 16 different polypeptides (Walker, JE and Collinson, TR (1994) FEBS
Lett.346: 39-43). Six of these polypeptides (subunit alpha,
The beta, gamma, delta, epsilon, and ATP inhibitor proteins IF 1) contain the globular catalytic F 1 ATP degrading enzyme portion of the complex, located outside the mitochondrial membrane. The remaining 10 polypeptides (subunits, a, b, c, d, e, f, g
, F6, OSCP, and A6L) contain the transmembrane F0 portion of the complex that mobilizes hydrogen ions. Like the other members of the respiratory chain, all but two of the polypeptide subunits of ATP synthase are nuclear gene products that are imported into mitochondria. Enzyme complexes similar to mammalian ATP synthase have been found in all cell types and chloroplasts and bacterial membranes. This universality indicates the central importance of this enzyme for ATP metabolism. Transcriptional regulation of these nuclear-encoded genes is considered to be the main means for controlling the biosynthesis of ATP synthase. There are multiple mitochondrial pathologies due to the important role of mitochondrial oxidative phosphorylation in cellular energy production, generation of reactive oxygen species and initiation of apoptosis. By this, it is clear that mitochondrial disease encompasses a set of clinical problems commonly associated with hearts, muscles, and kidneys and tissues with high energy requirements such as the endocrine system. Over the past 11 years, there has been a considerable body of evidence linking defects in mitochondrial energy generation pathways, oxidative phosphorylation, to a wide variety of degenerative diseases, including myopathy and cardiomyopathy. Most classes of pathogenic mitochondrial DNA mutations affect the heart in association with a variety of other clinical conditions that may include skeletal muscle, central nervous system (including the eye), endocrine system, and renal system. Nuclear mutations that cause mitochondrial disorders have been described. They are often found in highly conserved subunits. Mitochondrial disorders with nuclear mutations include myopathy (PEO, MNGIE, congenital muscular dystrophy, carnitine disorder), encephalopathy (Leigh's disease, infantile type, Wilson's disease, deafness-dystonia syndrome), other systemic disorders and Including cardiomyopathy.

The discovery of new ATP synthase subunits and polynucleotides encoding them, by providing new compositions that are useful in the diagnosis, prevention, and treatment of cancer myopathies, immune disorders, and neurological disorders. Answer the demand of the field. The protein of SEQ ID NO: 270, or a portion thereof, is believed to be involved in cellular respiration, preferably as the mitochondrial ATP synthase subunit. Preferred polypeptides of the invention have the SEQ ID NOs that have any of the biological activities described herein.
There are 270 fragments. The object of the present invention is to provide a composition for targeting a heterologous compound, either a polypeptide or a polynucleotide, to mitochondria by recombinantly or chemically fusing a fragment of the protein of the present invention to a heterologous polypeptide or polynucleotide. And how. Preferred fragments include, but are not limited to, a signal peptide, an amphipathic alpha helix and / or Herrman,
Neupert, and other fragments of proteins of the invention, including but not limited to matrix targeting signals defined in Curr, or parts thereof, including targeting signals for mitochondria. Opinion Microbiol. 3:21
0-4 (2000); Bhagwat et al., J. Biol. Chem. 274: 24014-22 (1999), Murphy Tr.
ends Biotechnol. 15: 326-30 (1997); Glaser et al., Plant Mol Biol. 38: 311-.
38 (1998); Ciminale et al., Oncogene 18: 4505-14 (1999). Such heterologous compounds can be used to modulate mitochondrial activity. For example, they can be used to induce and / or prevent mitochondria-induced apoptosis or necrosis. In addition, heterologous polynucleotides can be used in mitochondrial gene therapy for replacement of defective mitochondrial genes and / or inhibition of deleterious expression of mitochondrial genes. The invention further provides mitochondrial cytopathies, necrosis, aging, myopathy, cancer, and Alzheimer's disease, Huntington's chorea, Parkinson's disease, Tencan, Down's syndrome, dementia, multiple sclerosis, and amyotrophic lateral sclerosis. Proteins, or portions thereof, for the diagnosis, prevention, and / or treatment of some disorders in which the mitochondrial respiratory electron transport chain is impaired, including but not limited to neurodegenerative diseases such as Methods and compositions for use. For diagnostic purposes, expression of the proteins of the invention may be determined by Northern blotting, RT, as described herein.
-Can be studied using PCR, immunoblotting methods and compared to expression in control individuals. For prophylactic and / or therapeutic purposes, the proteins of the invention may be used to enhance electron transfer, as well as increase energy delivery, using any of the gene therapy methods described herein or known to those of skill in the art. Can be used.

In other embodiments, the present invention further employs Sjogren's syndrome, Addison's disease, bronchitis, dermatomyositis, using techniques known to those of skill in the art, including antisense or triple helices as described herein. Polymyositis, glomerulonephritis, diabetes, emphysema, Graves' disease, atrophic gastritis, lupus erythematosus, myasthenia gravis, multiple sclerosis, autoimmune thyroiditis, ulcerative colitis, anemia, pancreatitis, scleroderma, Some disorders in which the mitochondrial respiratory electron transport chain needs to be damaged, including but not limited to rheumatoid and osteoarthritis, allergic rhinitis, atopic dermatitis, dermatomyositis, polymyositis, and gout. The present invention relates to methods and compositions using the protein of the present invention or a part thereof for the diagnosis, prevention, and / or treatment of. In addition, antibodies to the proteins of the invention or portions thereof can be used to detect mitochondrial organelles and / or mitochondrial membranes for histological purposes using any of the techniques known to those of skill in the art. <Protein of SEQ ID NO: 271 (internal designation number 117-001-5-0-G3-CS)> The protein of SEQ ID NO: 271 is homologous to the family of lipopolysaccharide (LPS) binding proteins (LBP). Several families of proteins include L, which includes (a) lipopolysaccharide binding protein (LBP), and (b) bactericidal permeability enhancing protein (BPI).
Has the ability to bind PS. Cholesteryl ester transfer protein (CETP), which is involved in the transfer of insoluble cholesteryl ester in reverse cholesterol transport,
It shares some homology with members of the LPS-binding family of proteins. Lipopolysaccharide (LPS), also called endotoxin, is a major component of the outer membrane of Gram-negative bacteria. It consists of a core oligosaccharide and a serotype-specific O side chain polysaccharide that binds lipid A. LPS is a potent mediator of the inflammatory response, stimulating the expression of many proinflammatory and procoagulant compounds in monocytes, macrophages, and endothelial cells.
Although these responses are important in controlling and eliminating localized infections, LPS
Systemic exposure to elicit some adverse effects. These include (a) induction of the inflammatory cascade, (b) damage to the endothelium, (c) extensive coagulopathy, and (d) organ damage. Systemic exposure to LPS can result from direct infection with Gram-negative bacteria, resulting in complications of Gram-negative sepsis. Diseases associated with Gram-negative infection or endotoxemia include bacterial meningitis, neonatal sepsis, cystic fibrosis, inflammatory bowel disease, and cirrhosis, Gram-negative pneumonia, Gram-negative abdominal abscess, hemorrhagic shock , And disseminated intravascular coagulation. Subjects who are leukocytopenic or neutropenic, including subjects who have been treated with chemotherapy or immunocompromised, are particularly susceptible to bacterial infections and subsequent effects of endotoxin exposure.

Gram-negative sepsis remains one of the primary contributors to severe systemic inflammation in hospitalized and immunocompromised patients. Alternatively, changes in intestinal permeability due to various conditions, including trauma, may cause translocation of bacteria / LPS into the bloodstream. Bacteria translocated from the gut are immunosuppressed after surgery (Little et al., Surgery. 114: 87-91 (1993).
) And is believed to play a major role in hemorrhagic shock. Therefore, there is great interest in characterizing the proteins involved in the biological response to LPS, as well as finding therapeutics that can counter the effects of LPS in pathological situations. LBP is a 60 kDa glycoprotein synthesized in liver and is present in normal human serum. LBP expression is upregulated in response to infection, inflammation, and toxic mediators. LBP expression was induced in animals challenged with LPS, silver nitrate, terpentine, and Corynebacterium parvum (Geller et al., Surgery 128: 22-28 (1993); Gallay et al., Infect. Im
mun. 61: 378-383 (1993); Tobias et al., J. Exp. Med. 164: 77-793 (1986)).
LBP levels correlate with exposure to LPS and elevated levels (especially sustained elevated levels)
Associated with poor prognosis clinical outcome in patients with sepsis (US Pat. Nos. 5,484,705 and 5
, 804, 367, the entire text of which is incorporated herein by reference). A portion of the LBP molecule (N-terminal 1-197 amino acids) binds to the lipid A portion of the LPS molecule to form a high affinity LBP / LPS complex (Tobias et al., J. Biol. Chem. 264: 10867).
-10871 (1989)). The LBP / LPS complex, through its interaction with the monocyte differentiation antigen CD14,
Enhances cellular response to LPS (Wright et al. Science. 249: 1431-1433 (1990)
); Lee et al., J. Exp. Med. 175: 1697-1705 (1992)). LPS can be transferred from LBP to membrane bound or soluble CD14. Activated CD14 then interacts with endothelial cells,
It may induce an inflammatory response. The C-terminal portion of LBP is required to transfer LPS to CD14 (US Pat. No. 5,731,415; Theofan et al., J. Immunol. 152: 3624-29 (1994); H
an et al., J. Biol. Chem. 269: 8172-75 (1994)). Evidence also suggests that LBP can neutralize LPS by interacting with serum lipoproteins or internalizing the LBP / LPS / CD14 complex by neutrophils ((Wurfel et al., J. Exp. Med. 180: 1
025-1035 (1994); Wurfel et al., J. Exp. Med. 181: 1743-54 (1995); Gegner et al., J. Biol. Chem. 20: 5320-5325 (1995)).

The invention provides a polypeptide of SEQ ID NO: 271 and a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 271. In one embodiment, SEQ ID NO: 271
Polypeptide is compatible with the polypeptide encoded by the human cDNA which is clone 181-20-3-0-B5-CS. The present invention also includes bioactive fragments of the protein of SEQ ID NO: 271 and polynucleotide sequences encoding these bioactive fragments. In a preferred embodiment, the bioactive fragment of SEQ ID NO: 271 is encoded by clone 181-20-3-0-B5-CS, as well as the first encoded by clone 181-20-3-0-B5-CS. Contains 181 amino acids. A "biologically active fragment" is defined as a peptide or polypeptide fragment of SEQ ID NO: 271 that has at least one of the biological functions of a full-length protein (eg, the ability to bind bacterial LPS). The invention also provides a variant of SEQ ID NO: 271. These variants have at least about 80%, more preferably at least about 90%, even more preferably at least about 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 271. Variants according to the present invention may also have SEQ ID NO: 271 such as the biological functions described above.
It has at least one function or structural characteristic of. The present invention also provides bioactive fragments of variant proteins. Unless otherwise stated, the methods disclosed herein can be carried out using the polypeptide of SEQ ID NO: 271 or variants thereof. Similarly, the methods of the invention can be practiced with bioactive fragments of the protein of SEQ ID NO :, or variants of said bioactive fragments. Due to the redundancy of the genetic code, a variety of different DNA sequences can encode SEQ ID NO: 271. It is within the skill of one in the art to create these alternative DNA sequences that encode proteins having identical or substantially identical amino acid sequences. These mutant DNA sequences are therefore within the scope of the invention. As used herein, a "substantially identical" sequence is an amino acid substitution that does not substantially affect biological activity,
By a sequence with deletions, additions or insertions is meant. Fragments that retain one or more of the characteristic biological activities of SEQ ID NO: are also included in this definition. A "recombinant nucleotide variant" is an alternative polynucleotide that encodes a particular protein. They can be synthesized, for example, by taking advantage of the "redundancy" of the genetic code. Optimizes various codon substitutions, such as specific restriction sites or silent changes that produce codon usage-specific mutations, for cloning into plasmid or viral vectors, or expression in specific prokaryotic or eukaryotic host systems, respectively Can be introduced to

The protein of SEQ ID NO: 271 and variants thereof can be used to raise antibodies by methods known in the art. Antibodies can be monoclonal or polyclonal. Antibodies can also be synthesized by known methods against the fragment of SEQ ID NO: 271, as well as variants thereof. The invention also provides an antibody that specifically binds to the bioactive fragment of SEQ ID NO: 271 or a variant of SEQ ID NO: 271. The invention also provides immunoassays used to screen, monitor, or diagnose exposure to LPS. In one embodiment, the diagnostic assay is
LBP levels are measured in patient plasma samples. LBP levels are known to increase in response to exposure to LPS, and thus measuring protein levels of SEQ ID NO: 271 can provide early signs of Gram-negative infection or endotoxin exposure. The invention provides a method of treating an individual infected with a Gram-negative bacterium, comprising administering a therapeutically effective composition comprising SEQ ID NO: 271. In one embodiment, the protein lacks the C-terminal portion (or part of the C-terminal domain) required to transfer LPS to CD14. LPS is trapped by excess N-terminal fragments making it incapable of inducing an inflammatory response (see, eg, US Pat. No. 5,731,415, the entire text of which is incorporated herein by reference). Another aspect of the invention provides a method for prophylaxis. This method treats an individual by administering a therapeutically effective amount of a composition comprising SEQ ID NO: 271. Examples in which this aspect of the invention may be practiced include, but are not limited to, conditions associated with increased translocation of intestinal bacteria and endotoxin, particularly prior to surgery. In addition, patients at high risk for latent Gram Gram infections, including but not limited to patients undergoing chemotherapy or immunocompromised (eg, AIDS), would benefit from such treatment. Can receive. Such applications are described in US Pat. No. 5,990,082, the entire text of which is incorporated herein by reference. The N-terminal portion of LBP, which lacks the ability to induce an inflammatory response, increases the association of these molecules with LPS and other proteins or other proteins that can help eliminate endotoxin from patients exposed to Gram-negative bacteria. It can be fused to a fragment (bactericidal / permeability-increasing protein or BPI). Such preparations are incorporated by reference in their entirety for each publication, the following patents: International Patent Application Publication WO 95/19179 A,
It can be used to treat and control some Gram-negative, Gram-positive, or fungal infections, as described in WO 95/19180 A, WO 95/19372 A and WO 96/34873 A.

The present invention also provides a method of removing endotoxin from recombinantly produced proteins. In one embodiment, the recombinantly produced protein is obtained from Gram-negative bacteria. In a preferred embodiment, the bacterium is E. coli. In other embodiments, the protein of SEQ ID NO: 271, a bioactive fragment, variant, or derivative thereof is contacted with a composition comprising a recombinantly produced protein. The contacting step occurs between SEQ ID NO: 271 immobilized on the substrate or SEQ ID NO: 271 present in the free solution. In addition, the protein of SEQ ID NO: 271, a bioactive fragment thereof, or a derivative thereof can be used in a diagnostic assay to measure LPS levels in patient plasma samples. In such an assay, a serum sample is bound to a solid matrix such as a membrane, plastic, treated plastic, or other support and then cloned with the protein of SEQ ID NO: 271. Visualization can be achieved by fusing the protein of SEQ ID NO: with some enzymes and then treating with chromogenic, fluorogenic, luminescent substrates. Alternatively, the protein of SEQ ID NO: 271, a biologically active fragment, variant, or derivative thereof can be attached to a fluorescent or luminescent protein or compound. The linkage may be chemical or it may be produced by recombinant techniques known to those skilled in the art. In addition, SEQ ID NO: 271
Antibodies raised against the protein of E. coli, biologically active fragments, variants, or derivatives thereof can be used to visualize the LPS / protein 271 complex using immunoassays known to those of skill in the art. <Protein of SEQ ID NO: 266 (Internal Designation Number 116-110-2-0-F4-CS)> The protein of SEQ ID NO: 266, which is highly expressed in the testis, is encoded by the cDNA of SEQ ID NO: 25 and contains about 100 amino acids. Ly-6 family of GPI-coupled cell surface glycoproteins (PS00 consisting of one or more copies of a conserved domain of residues
983; LY6_UPAR). The protein of SEQ ID NO: 266 shows significant structural similarity to mouse Ly-6 antigen, human CD59 and herpesvirus CD59 homolog. The protein of SEQ ID NO: 266 presents one copy of the u-PAR / Ly-6 domain motif, in which all 10 extracellular cysteine residues are conserved. The mature protein sequence contains a relatively high proportion of cysteine residues (10/105), suggesting that multiple disulfide bonds stabilize its tertiary structure. In addition, the 124 amino acid long protein of SEQ ID NO: 266 is
It has a size similar to many members of the Ly-6 family. In addition, the proteins of the invention have predicted signal peptide structures (positions 1-19) and CPI in the membrane.
It has the C-terminal hydrophobic fragment (positions 101-121) required for anchoring. Thus, the proteins of the invention have a clear evolutionary relationship with the Ly-6 / uPAR family, especially the Ly-6 subfamily.

Ly-6 / uPAR protein family members share one or several repeat units of the Ly-6 / uPAR domain, defined by a unique disulfide bond between 8 or 10 cysteine residues. This family can be divided into two subfamilies. One contains a GPI-anchored glycoprotein receptor with 10 cysteine residues. The other subfamily contains a secretory single domain, which is the snake and frog cytotoxin, whose members generally differ in that they have 8 cysteines and no GPI anchoring signal sequence ( A
Ndermann K, et al., Protein Sci 8 (4): 810-819 (1999)). Ly-6 family members are low molecular weight phosphatidylinositol anchor glycoproteins that have significant amino acid homology through a unique cysteine-enriched protein domain that is primarily associated with O-linked carbohydrates. Their GPI binding is required to anchor these cell surface proteins outside the lipid bilayer membrane. The Ly-6 family includes human CD59, squid Sgp1 and Sgp2, urokinase plasminogen activator receptor, mouse Sca-1 and Sca-2, and many other proteins that protect against complement-mediated membrane damage. The general structure observed in the Ly-6 family is urokinase-type plasminogen activator and alpha- from snake venom.
Similar in structure to the neurotoxin receptor (Fleming TJ et al., J Immunol 150: 5379-5390.
(1993); Ploug M and V Ellis FEBS Lett 349: 163-168 (1994)). Ly-6 cell surface proteins are specifically expressed in several hematopoietic lineages and appear to function in signal transduction and cell activation primarily in mouse lymphocytes. Analysis using anti-Ly-6A / E monoclonal antibody also revealed that Ly-6 in brain tissue
In situ expression of the molecule was demonstrated (staining was primarily associated with vascular elements throughout the brain). These proteins do not appear to be expressed during embryonic or neonatal development (Cray C et al. Brain Res Mol Brain Res 8 (1): 9-15 (1
990)). Ly-6 protein expression has been shown to be factor dependent. For example, Ly-6A / E expression, which normally occurs in hematopoietic stem cells, fibroblasts, and T and B lymphocytes, has been shown to be massively induced by IFN-s in a variety of tissues and cell lines. ing. In addition, Ly-6E Ag associates with tyrosine kinases in T cells, and reduced Ly-6E expression in T cells impairs normal functional response as well as tyrosine kinase activity in these cells. In addition, IFN is a memory C
Important for D8 + T cell production, Ly-6C Ag expression has been demonstrated to be a strong marker of memory phenotype (Mehran M. et al., Journal of Immunology 163.
811-819 (1999)). Similar to its mouse counterpart, human homologue of Ly-6 gene, 980
Four genes are responsive to IFN. The 9804 gene is also inducible by retinoic acid during the differentiation of acute promyelocytic leukemia cells. Furthermore, cultured glial and neuronal cells express high levels of Ly-6A / E after incubation with cytokines, including rIFN-gamma. (Cray C et al., Brain Res Mol Brain Res. 8 (1
): 9-15 (1990)). Human protein RoBo, another member of the Ly-6 family
-1 shows increased expression in response to two modulators of bone metabolism, estradiol and intermittent mechanical load, suggesting its involvement in bone homeostasis (Noel LS et al., J Biol Chem, Vol. 273). (7): 3878-3883 (1998)). Such factor dependence of expression causes the Ly-6 protein to be candidates or targets for allogeneic responses and autoimmune diseases. For example, high levels of factor-induced expression of LY-6 are associated with nephritic lupus (Blake PG et al., J Am Soc Nephrol 4: 1140-1150 (1993)).
.

The murine Ly-6 molecule has an interesting pattern of tissue expression during hematopoiesis from pluripotent stem cells to lineage progenitor cells and in specific leukocyte subpopulations of peripheral lymphoid tissue. These patterns suggest a close link between regulation of Ly-6 expression and development and homeostasis of the immune system (Gumley TP et al., Immunol Cell Biol 73 (4): 277-296).
(1995)). Ly-6M messenger RNA (mRNA) is readily detectable in hematopoietic tissues (bone marrow, spleen, thymus, peritoneal macrophages), and kidney and lung (Patterson JM et al. Blood 95 (10): 3125-3132 (2000). )). Normally, human blood cells are protected from self-complement activation by membrane proteins that block the assembly of functional complement pores. One such protein is human Ly-6 CD59. Administration of CD59 prevents hemolytic disease or thrombosis. In addition, the CD59 protein protects against complement-mediated lysis and endothelial cell activation leading to acute rejection, and thus can be administered during xenotransplantation (Binette, JP
And Binette, MB, Scanning Microcs., 7: 1107-10 (1993)). The surface receptor for urokinase plasminogen activator (uPAR) has recently been recognized as a key molecule in controlling plasminogen-mediated extracellular proteolysis. Surface plasminogen activation regulates the binding between cells, basement membranes and extracellular matrix, and consequently the ability of cells to migrate and enter adjacent tissues (Roldan AL et al., EMBO J 9 (2 ): 467-474 (1990)). Certain factors of the PA system, such as u-PAR, have been detected within male reproductive tract organs in a variety of species. Morphological studies provide support for the involvement of the PA system in human male reproductive physiology (Gunnarsson M et al. Mol Hum Reprod 5 (10): 934-940 (
1999)). It has been suggested that LY-6 protein plays an important role in disorders such as cancer, nephropathy, autoimmune disease, hemolytic disease, thrombosis, Alzheimer's disease and the like. Several members of the murine Ly-6 supergene family have been shown to be involved in the progression of certain mouse tumors, due to higher expression levels in high-grade cells than in tumor cells with a low-grade phenotype. Are involved. Sorting of tumor cells into high or low Ly-6E.1 expression subpopulations by flow cytometry
We gave rise to cells expressing a high-grade or low-grade phenotype respectively. Furthermore, LY-6
Has been shown to be highly expressed in non-lymphoid tumor cells from various tissues in mice. Upregulation or high expression correlates with a more aggressive phenotype and causes more efficient local tumor production (Katz et al., Int J Cancer
59: 684-91 (1994)).

Cells from angiogenic tumors express a higher tumorigenicity phenotype and a higher capacity to give rise to artificial lung metastases compared to cells from tumors with poor angiogenesis. These cells also express significantly higher levels of the lymphocyte activating protein Ly-6E, so their angiogenic phenotype appears to be co-regulated by Ly-6 (Sagi-Assif O et al. , Immunol Lett 54 (2-3): 207-13 (1996)). Several LY-6 proteins block the secretion of interleukin II (IL-2), a licensed anti-cancer agent and a major regulatory hormone in cell-mediated immunity (Fleming
TJ and TR Malek. J Immunol. 153: 1955-62 (1994)). IL-2 stimulates the proliferation of both T and natural killer cells and also activates NK cells that can directly lyse freshly isolated, solid tumor cells. High-grade tumors, high Ly-6E.1-expressing cells also express high levels of the receptor for urokinase plasminogen activator (uPAR), but low-grade tumors, low
Ly-6E.1 expressing cells also express low levels of uPAR. Transfection studies have shown that it is by chance involved in conferring a highly aggressive phenotype on tumor cells that express high levels of Ly-6E.1. E48, a human homologue of mouse ThB Ly-6 protein, is expressed in head and neck squamous cell carcinoma cells. In E48-stimulated cells, binding of E48 to its microenvironmental ligand appears to transduce a signal that upregulates expression of the FX enzyme in these cells, leading to increased levels of GDP-L-fucose (Rinat Eshel Et al., J Biol Chem, Vol. 275 (17): 12833-12840 (20
00)). A congenital disorder called LADII, in which leukocytes adhere to the vascular endothelium, represents a generalized fucose deficiency and a major defect in leukocyte intracellular transport and function.
Ly-6-deficient mutants of mouse tumors exhibit modified uptake of fucose and mannose into cellular glycoconjugates (Witz IP J. Cell. Biochem. Suppl. 34: 61-66.
(2000)). The protein of SEQ ID NO: 266 is a new member of the Ly-6 protein family,
Thus, it is a specific cell surface glycoprotein antigen involved in signal transduction and cell activation, proliferation and differentiation. A preferred polypeptide of the invention is a polypeptide comprising the amino acids of SEQ ID NO: 266 from positions 1-18 and 19-124. Other preferred polypeptides of the present invention are fragments of SEQ ID NO: 266 having any of the biological activities described herein.

In one embodiment, the invention uses a protein of the invention or a portion thereof as a marker protein for selectively identifying a tissue, preferably testis,
Methods and compositions. For example, the proteins of the invention or portions thereof can be used to synthesize specific antibodies using techniques known to those of skill in the art. Such tissue-specific antibodies can then identify tissues of unknown origin, such as differentiated tumor tissues that have metastasized to forensic samples, different body sites, etc., or use immunochemistry to identify different tissue types in tissue cross-sections. Can be used to classify. Other embodiments of the invention use the proteins of the invention or portions thereof and related compounds and derivatives to diagnose developmental and malignant disorders of tissues, including urogenital tissues and other tissues of the reproductive system of both sexes. Regarding the method. For example, a biological sample is taken from a cancer patient or at risk of developing cancer to detect the level of the polynucleotide of SEQ ID NO: 25 or the encoded polypeptide in the cells of the sample. Detection of elevated levels of SEQ ID NO: 25 polynucleotide or encoded polypeptide in the sample relative to control levels is indicative of the presence of malignant cells in the patient. Expression of the proteins of the invention can be carried out by Northern blotting, RT-P
It can be studied using any of several methods, including but not limited to CR or immunoblotting. Another embodiment of the present invention is the use of a protein of the present invention or a portion thereof in recombinant protein form as a pharmaceutical in the treatment of developmental and malignant disorders in tissues including genitourinary tissues and other tissues of the human reproductive system. Compositions and methods for use. In particular, the proteins of the invention or parts thereof can be used for the treatment of disorders having signs of male infertility. In another embodiment of the invention, an antibody that binds to a protein of the invention, or a portion thereof, is an approved anti-cancer agent in the treatment of tumors, such as human genitourinary tumors, and in cell-mediated immunity. Used to enhance the secretion of interleukin II, the major regulatory hormone. Such antibodies can be used alone or in combination with a substance capable of exfoliating or killing cells as a treatment for genitourinary disorders or cancers in which the protein of the present invention is overexpressed. The proteins of the invention or portions thereof also include various forms of cancer such as genitourinary cancer, cancer, sarcoma, atherosclerosis, angiogenesis, and benign tumors.
It can be used to treat diseases that may require transplantation. As mentioned above, the Ly-6 family includes several proteins that are similar to the proteins of the invention and are capable of protecting cells from complement-mediated membrane damage. Therefore, in another embodiment of the invention, the recombinant protein encoded by SEQ ID NO: 25 or a fragment thereof is used to prevent complement-mediated lysis that normally causes acute rejection and to block endothelial cell activation. Administered during xenotransplantation.

Furthermore, prevention of complement-mediated lysis is of particular importance in reproductive therapy in humans and animals, where functional survival of embryonic cells during in vitro handling is essential.
Sperm storage is of widespread importance in commercial animal breeding programs, human sperm donor programs, and treatment of certain disease states. For example, for men diagnosed with cancer or a disease that can ultimately interfere with sperm production, and for the purpose of assisting reproduction, in which sperm are stored for use elsewhere or at a time. The sperm sample can be frozen. The procedure used in such cases is to wash the sperm sample to separate the sperm enriched fraction from the non-sperm components of the sample, such as semen plasma or debris; and in dead sperm and ejaculate. Isolating healthy, motile sperm from white blood cells; freezing or refrigerating for later use or shipping to females at different locations; culture in diagnostic tests, or in vitro fertilization or intracytoplasmic sperm Spreading or diluting sperm for use in therapeutic interventions such as infusion (Cohen et al., 12: 994-1001 (1997)). Once the sperm have been washed or isolated, they are then spread (or diluted) in culture or the medium is stored for various uses (sperm analysis, diagnostic tests, assisted reproduction). These uses of extended or diluted sperm require a formulation somewhat different from the basal medium (
For a review, see US Pat. No. 6,140,121, Ellington et al., Oct. 2000); however, sperm survival is suboptimal outside the female reproductive tract in all cases. New additions to the diluent or storage medium that can improve functional preservation of sperm are useful. Therefore, in another preferred embodiment of the present invention, the purified recombinant protein encoded by SEQ ID NO: 25 or a fragment thereof can be added as a component of a pharmaceutical medium designed for sperm protection. The methods used to construct such storage media are generally known to those of skill in the art (eg, Oliver SA. Et al., US Patent 5,897,987, April 1999; Cohen J. et al., Supra). . Conversely, in yet another embodiment of the present invention, a ligand, inhibitor, neutralizing antibody or other biological agent that recognizes and binds to and blocks the protein of the present invention is for male contraceptive purposes. It can be used as a constituent of a designed pharmaceutical formulation. In yet another embodiment of the invention, a chimeric ligand or derivative that recognizes a protein of the invention or a portion thereof and is internalized in a cell is a urogenital or other tissue expressing the protein of SEQ ID NO: 266. It can be used to design drug delivery systems that target delicately. For example, such a recognition molecule can be incorporated into the liposome membrane to allow specific delivery of the liposome to cells expressing the protein of SEQ ID NO: 266. Methods of designing such drug delivery systems are known to those of skill in the art (Smith HJ Introduction to the principles of drug design and action).
Third edition (1998)).

<Proteins of SEQ ID NOs: 417, 413, 418 (Internal Designation No. 188-45-1-0-D3-CS, 18
8-26-4-0-F5-CS, and 188-5-1-0-H6-CS)> SEQ ID NOS: 417, 41 encoded by the cDNAs of SEQ ID NOS: 176, 172, and 177
Proteins 3 and 418 are expressed in the brain and exhibit strong homology with proteins having redox activity (eg Genbank accession numbers: AK001293 and AF0296).
89, and Geneseqp accession number: Y59180). The protein of SEQ ID NO: 418 (320 amino acids) is a variant of AK001293 (322 amino acids). AK001293 has 6 extra nucleotides in the same ORF as SEQ ID NO: 418 and produces a longer protein. SEQ ID NO: 418 is the Pf from position 16-313
am Presents the zinc-binding dehydrogenase (adh zinc) signature. SEQ ID NO: 418 presents all conserved residues in the motif, except for histidine, which is believed to be the zinc ligand. This lack of zinc ligand residues is characteristic of quinone oxidoreductase (QOR), a subfamily of zinc-binding dehydrogenases. SEQ ID NO: 413 (191 amino acids) shares the first 172 amino acids with SEQ ID NO: 418. However, the deletion of one nucleotide at position 583 (corresponding to amino acid 173) in the cDNA of SEQ ID NO: 413 creates an ORF change compared to SEQ ID NO: 418 and AK001293. SEQ ID NO: 417 is a short protein (20 amino acids) whose sequence corresponds to the N-terminal part of other proteins of the invention. The presence of a T at position 128 on the cDNA (instead of the published sequence and G in SEQ ID NOs: 413 and 418) creates a stop codon and thus a short protein. SEQ ID NOS: 417, 413, and 418 are QOR, a family of zinc-binding dehydrogenases.
Is similar to. QOR is a cytoplasmic redox-regulated flavin enzyme that catalyzes one or two electron reduction of quinones. QOR binds NADP as well as is inhibited by dicoumarol. QOR activity protects cells against toxicity, mutagenicity, and cancer due to exposure to the environment and synthetic quinones and their precursors. Therefore, QOP plays a central role in monitoring cellular redox status and acts to protect against oxidative stress induced by various metabolic contexts (Raina AK et al., (1999).
) Redox Rep. 4: 23-7). Redox enzyme activity also allows activation of bioreductive anti-cancer agents (Begleiter A. et al. (1996) Br. J. Cancer Suppl. 27: S9-14).
.

Quinone metabolism involves the enzymatic reduction of quinones with one or two electrons. Upon activation of a quinone-containing anti-tumor agent, this reduction results in the formation of the anti-cancer agent semiquinone or hydroquinone. As a result of these enzymatic reductions, semiquinones donate their excess electrons to oxygen, forming superoxide radical anions and native quinones. These reductions by reductases, followed by oxidation by molecular oxygen (diatomic oxygen), known as redox cycling, continue until the system is anaerobic. In the case of two-electron reduction, hydroquinone is stabilized,
As a result, it is excreted by the organism in the detoxification pathway. The antioxidant response of cells is mediated by a set of detoxification / protection protein devices. The promoters of the genes encoding these proteins contain a common cis element called the antioxidant response element (ARE). Nrf, Jun, Fos, Fra
, Maf, YABP, ARE-BP1, Ah (Aromatic Hydrocarbon) Receptors, Multiple Transcription Factors, and Estrogen Receptors
Bind to ARE. Among these factors, the Nrf-Jun heterodimer positively regulates gene induction in ARE-mediated expression and response to antioxidants and xenobiotics (Dhakshinamoorthy S. et al. (2000) Curr Top Cell Regul. 36: 201-16). On the other hand, c-Fos suppresses ARE-mediated gene expression (Venugopal,
R. and Jaiswal, AK (1996) Proc. Natl. Acad. Sci. USA 93, 14960-5). Elevated levels of QOR activity have been reported in several types of tumors such as liver, colon, lung and breast (Belinsky M., Jaiswal AK, (1993) Cancer Meta.
stasis Rev 12: 103-17). Bioreactive antitumor agents are an important class of anticancer agents that require activation by reduction. For this reason, QOR may be the underlying target for the development of new antitumor compounds. The specific QOR is already mitomycin C
, Metabolism, activation and mechanism of cytotoxicity of some anti-cancer agents such as indoloquinone E09 (Ross D. et al. (1994) Oncol. Res. 6: 493-500), CB 1954.
(Knox RJ et al. (2000) Cancer Res. 60: 4179-86), or anti-estrogen in breast cancer (Montano MM, Katzenellenbogen BS (1997) PNAS. 94:25.
81-6). Furthermore, some of the QOR family proteins are believed to be involved in preventing apoptosis after oxidative stress. The tumor suppressor gene p53
Directly involved in the induction of apoptosis in dividing cells and hippocampal pyramidal neurons (Jordan J. et al. (1997) J. Neurosci. 17: 1397-405), the QOR gene
53 described as a regulatory gene ((Kostic C, Shaw PH (2000) Oncogene 1
9: 3978-87).

The proteins of SEQ ID NOs: 417, 413 and 418 are believed to have redox activity, probably as a QOR. Therefore, they are predicted to act as endogenous antioxidants against oxidative stress, possibly using NADP as a cofactor. The protein of the present invention can be used for inactivating toxins and activating bioreductive anticancer agents. Moreover, they prevent apoptosis after oxidative stress and can be regulated by p53. The proteins of SEQ ID NOS: 417 and 413, in contrast to SEQ ID NO: 418, do not contain Pfam zinc-binding dehydrogenase (adh zinc), so they are classified as competitive inhibitors of functional proteins, the dominant negative form. To work. The oxidoreductase activity of the proteins of the invention can be assayed using any of the techniques known to those of skill in the art. For example, the measurement of the rate of oxidation and oxygen consumption of NADPH, and the detection of semiquinone and reactive oxygen species are described by Gutierrez PL (Gutierrez PL (2000
) Front. Biosci. 5: D629-38) or by methods known to those skilled in the art. The enzymatic activity of the proteins of the invention in specifically affected and control tissues can be assayed by histochemical staining. In vitro and in vivo assays can be performed to confirm the role of the protein in cellular antioxidant response. Transcriptional levels of the genes encoding the proteins of the invention were determined by exposure to quinone or quinone-derived compounds such as beta-naphthoflavin (beta-NF), as described by Belinsky M. and Jaiswal AK (supra). Later and in response to transcription factors such as as Nerf, Jun and c-Fos, or in the presence of p53, can be measured using standard methods. In one embodiment of the invention, the invention can be used to detect specific cell types in vitro or in vivo. For example, since the present protein is overexpressed in the brain, a reagent that can specifically recognize the present protein can be used as a marker for brain cells. Brain-specific markers identify specific tissues for tissue analysis,
And has several uses, including detecting the origin of tumor cells. Furthermore, the expression of this protein is expressed by Nrf, Jun, Fos, Fra, Maf, YABP, ARE-BP1,
Reagents specific for the detection of this protein also appear to be induced by transcription factors such as the Ah (aromatic hydrocarbon) and estrogen receptors, and p53, so that these proteins may also be detected in vitro or in vivo. Can be used as a marker for the activity of. Given the association between many of these proteins and diseases such as cancer, the ability to detect the presence or absence of this protein provides a powerful tool for disease diagnosis and screening. For any of these applications, expression of the protein can be detected using standard methods including Northern blot, Western blot, in situ hybridization, PCR and the like.

In another embodiment, the proteins of the invention serve as markers for oxidative stress of cells in vivo and in vitro. Thus, the proteins of the invention, or portions thereof, may be used in a wide variety of types of cancer as well as Alzheimer's disease (AD
), Amyotrophic lateral sclerosis (ALS) or neurodegenerative disorders such as Parkinson's disease (PD), and may be useful in the diagnosis of disorders related to oxidative stress. For diagnostic purposes, expression of the proteins of the invention may be determined, for example, by Northern blotting, RT-PCR,
It can be studied using immunoblotting methods and compared to expression in control individuals. Elevated protein levels of the invention in patients as compared to controls is
It shows a large shift in redox equilibrium and is thus indicative of the disease or susceptibility to the disease. The invention further relates to methods and compositions using the proteins of the invention or portions thereof for the prevention and / or treatment of disorders associated with oxidative stress, including those described above. For these purposes, the protein itself, or a polynucleotide encoding the protein, or an activator of protein expression, in a patient, or an unaffected individual with increased susceptibility to one of these disorders. May be administered to. In other embodiments, the proteins of the invention or portions thereof are used to prevent cells from undergoing apoptosis. Therefore, they are immunodeficiency syndromes (including AIDS), type 1 diabetes, pathogenic infections, cardiovascular and nerve injury, alopecia, aging, degenerative diseases including AD and PD, dystonia, label inheritance. Useful in the diagnosis, treatment and / or prevention of disorders and processes in which apoptosis is detrimental, including but not limited to optic atrophy and schizophrenia.
For all such diagnostic purposes, expression of the proteins of the invention can be studied using Northern blotting, RT-PCR, immunoblotting methods described herein and compared to expression in control individuals. . The present invention relates to methods and compositions using the protein of the present invention or a part thereof as a detoxifying enzyme for quinones. There are various quinones that have toxic effects in cells (eg, quinones derived from the oxidation of phenol metabolites of benzene, DA
Quinone, or menadione). Thus, the proteins of the invention, or portions thereof, are protective against the hematological and carcinogenic effects of benzene and benzene-induced diseases such as cancer, aplastic anemia, and pancytopenia. Furthermore, they detoxify DA quinones in the brain, thereby providing neuroprotection in Parkinson's disease. In yet another embodiment, the proteins of the invention, or portions thereof, are capable of protecting cells against menadione-induced oxidative stress with known effects on cardiomyocytes (Floreani M. et al. (2000). Biochem Pharmacol. 60: 601-
Five). For prophylactic and / or therapeutic purposes, the protein itself, or a polynucleotide encoding the protein, or an activator of protein expression can be administered.

In other embodiments, the protein may be targeted for chemotherapy specific to different types of cancer in order to ensure a favorable response to anti-cancer agents. In particular, the proteins of the invention or parts thereof can be used as activators of quinone family cytotoxic prodrugs. Therefore, the protein of the present invention or a part thereof can be administered to a patient in combination with an anti-cancer agent to activate the bioreductive anti-cancer agent. This co-administration may be co-administration, such as a mixture of oxidoreductase and drug, or may be separate and simultaneous or sequential. Cancer-specific antitumor agents based on QOR substrates can be designed as described by Xing J. et al. (Xing J. et al. (2000) Med.
Chem. 43: 457-66) and Li B. et al. (Li B et al. (1999) Chem. Res. Toxicol. 12: 1042-9). Alternatively, since the protein may be overexpressed in tumor cells, such a method would simply detect the level of the protein in the tumor cells and detect elevated levels of the protein in the tumor cells. This can be done by specifically administering the prodrug to the patient. <Proteins of SEQ ID NOs: 415, 310, 317 (internal designation number 188-29-2-0-H1-CS, 18
8-18-4-0-A9-CS, 188-9-2-0-E1-CS)> Mammalian inositol hexakisphosphate kinase 2 (IP6K2), an enzyme of the inositol phosphate pathway, has been cloned and Explained by two independent groups <Saiardi, A .; Erdument-Bromage, H .; Snowman, AM; Temps
t, P .; and Snyder, SH, (1999) Current Biology 9, 1323-1326, and
Katai, K .; Miyamoto, KI .; Kishida, S .; Segawa, H .; Nii, T .; Tanak
a, H .; Tani, Y .; Arai, H .; Tatsumi, S .; Morita, K .; Taketani, Y .;
And Takeda, E. (1999) Biochem. J. 343, 705-712>. The newly identified consensus sequence for inositol-polyphosphate kinase is <LV>-<LA>-<DE> -X (3-8)-
PX- <VAI>-<ML> -DXK- <ML> G <Saiardi, A .; Erdumen
t-Bromage, H .; Snowman, AM; Tempst, P .; and Snyder, SH (1999
) Current Biology 9, 1323-1326>. IP6K2 is InsP6 or Ins (1,3,4,5,6) P
Catalyzes the transfer of phosphate groups from 5 (substrates) to other proteins or small molecules such as nucleoside diphosphates. The invention provides the polypeptides of SEQ ID NOs: 415, 310 and 317 encoded by the cDNAs of SEQ ID NOs: 174, 69 and 76, respectively. The invention also provides bioactive fragments of SEQ ID NOs: 415, 310 and 317. In one embodiment, the polypeptides of SEQ ID NOs: 415, 310 and 317 are clones 188-29-2-0-H1-CS,
188-18-4-0-A9-CS, or 188-9-2-0-E1-CS is compatible with the corresponding polypeptide encoded by the human cDNA. A "bioactive fragment" is defined as a peptide or polypeptide fragment that has at least one of the biological functions (eg, kinase activity) of a full-length protein. Compositions of proteins / polypeptides of SEQ ID NOs: 415, 310, or 317 or biologically active fragments thereof are also provided by the present invention. These compositions can be made according to methods known in the art.

The present invention also provides variants of the proteins of SEQ ID NO: 415, 310, or 317. These variants have at least about 80%, preferably at least about 90%, and most preferably at least about 95% amino acid sequence identity to the amino acids encoded by SEQ ID NOs: 415, 310, or 317. . Variants according to the invention also have at least one functional or structural characteristic of the protein of SEQ ID NO: 415, 310, or 317. The present invention also provides bioactive fragments of variant proteins. Compositions of variants, or bioactive fragments thereof, are also provided by the present invention. These compositions can be made according to methods known in the art. Unless otherwise stated, the methods disclosed herein are SEQ ID NOs: 415, 310.
, Or a protein encoded by 317, a bioactive fragment of SEQ ID NO: 415, 310, or 317, a variant of SEQ ID NO: 415, 310, or 317, and a bioactive fragment of said variant. Due to the redundancy of the genetic code, a variety of different DNA sequences have been identified in SEQ ID NOs: 415, 310,
Alternatively, it can encode the 317 amino acid sequence. In a preferred embodiment, SEQ ID NO: 415
, 310, or 317 are clones 188-29-2-0-H1-CS, 188-18-4-0-A9-CS, or 1
88-9-2-0-E1-CS, or encoded by the cDNA of SEQ ID NO: 174, 69, or 76. It is within the skill of one in the art to create these alternative DNA sequences that encode proteins having identical or substantially identical amino acid sequences. These mutant DNA sequences are therefore within the scope of the invention. As used herein, a "substantially identical" sequence means a sequence having amino acid substitutions, deletions, additions or insertions that does not substantially affect biological activity. Also included in this definition are fragments that retain one or more characteristic biological activities of the protein encoded by SEQ ID NO: 415, 310, or 317. In one aspect of the invention, SEQ ID NOs: 415, 310, or 317 and variants thereof can be used for polyclonal or monoclonal antibody production. Sequence number
Both 415, 310, or 317 bioactive and immunogenic fragments, or variant proteins can be used for antibody production. Polyclonal and / or monoclonal antibodies can be produced by methods known to those of skill in the art.
The antibodies produced in accordance with the present invention can be used in various detection assays known to those of skill in the art. The antibody can be used to agonize or antagonize the biological activity of the protein of SEQ ID NO: 415, 310, or 317.

The proteins of SEQ ID NOs: 415, 310, or 317 are nucleoside triphosphates (NTPs)
It can be used for the synthesis of compounds. In one embodiment, the NTP compound produced is ATP
, GTP, CTP, or TTP. In this aspect of the invention, SEQ ID NOs: 415, 310,
Or 317 removes the phosphate from InsP6 or Ins (1,3,4,5,6) P5 and transfers it to a nucleoside diphosphate (eg ADP, CTP, GDP, or TDP), Create NTP. Conditions and methods for the synthesis of NTP compounds such as ATP are known to those of skill in the art. Therefore, the proteins of SEQ ID NOs: 415, 310, or 317 have industrially useful functions for the synthesis of commercially valuable products. The present invention also provides InsP6 or Ins (1,3,4,4,
5,6) Provide a method to measure the relative amount of P5. In this aspect of the invention, SEQ ID NO: 4
15, 310, or 317 were labeled with InsP6 or I by known kinase activity assays.
It can be used to transfer the phosphate group from ns (1,3,4,5,6) P5 to the acceptor substrate. <Protein of SEQ ID NO: 294 (internal designation number 181-16-2-0-A7-CS)> The protein of SEQ ID NO: 294 is encoded by the cDNA of SEQ ID NO: 53. It is to be understood that all of the features and uses of the polypeptide of SEQ ID NO: 294 described throughout this application also relate to the polypeptide encoded by the human cDNA which is clone 181-16-2-0-A7-CS. Let's do it. In addition, SEQ ID NOs described throughout this application
It will be appreciated that all of the 53 nucleic acid features as well as the uses also relate to the human cDNA clone 181-16-2-0-A7-CS. The gene has been isolated from fetal liver and its expression has also been detected in fetal kidney, placenta, liver, brain, hypertrophic prostate gland, salivary gland and testis. The data from WO 98/23435 is mainly expressed in bone marrow cell lines and to a lesser extent in human endometrial stromal cells, human adult small intestine and human pancreatic tumors. International Patent Application Publication WO 99/14484 discloses that slight expression was observed in the gastrointestinal system (0.227
), The reproductive system (0.193), and the hematopoietic / immune system (0.168). Ultimately, the protein was isolated from CD34 + cells and was derived from a hematopoietic lineage, including HL6 (granulocytes), Jurkat (T-lymphocytes), K562 (red / megakaryocytes) and U937 (mononuclear cells). It is also 55% identical and 76% similar to the CGI-128 protein found in cell lines.

Supernatants collected from cells expressing the product of this gene have been shown to increase the permeability of renal mesangial cell plasma membranes to calcium. Thus, the product of this gene is implicated in the activation of signaling pathways when it binds to receptors on the plasma membrane surface of mesangial cells and other cell types in addition to other cell lines or tissue cell types. It is considered. Thus, the polynucleotides and polypeptides include, but are not limited to, activating mesangial cells by contacting the cells with a full-length polypeptide or a polypeptide fragment that demonstrates this biological activity. Have. Furthermore, the present polynucleotides and polypeptides are incorporated by reference in their entirety in WO9915652.
Can be used in the method described in. Binding of ligands to receptors is known to modify intracellular levels of small molecules such as calcium, potassium, sodium, and pH and membrane potential. Small molecule concentration modifications can be measured to identify supernatants that bind to receptors on specific cells. Furthermore, when tested on a fibroblast cell line, the supernatant removed from cells containing this gene activated the EGR1 (Early Growth Response Gene 1) promoter element. Therefore, this gene is probably
Activates fibroblasts via the EGR1 signaling pathway. EGR1 is a signal transduction pathway different from Jak-STAT, and a gene containing the EGR1 promoter is induced in various tissues and cell types when activated, and leads to cell differentiation and proliferation (
International patent application publication WO 98/23435). A polynucleotide containing a sequence encoding the signal peptide of the present protein, for example, VLWLSGLSEPGAA / RQ, can be used to prepare a secretion vector. These vectors then facilitate the secretion of the fusion protein into the culture medium of cells that have been transfected with the construct of interest. Antibodies that specifically bind the signal peptide can be used to purify the fusion protein from this culture medium, if desired. The polynucleotides and polypeptides of the present invention can be used to differentially identify tissue (s) or cell type (s) present in a biological sample, as well as endothelial, mucosal, or epithelial cell damage, as well as hematopoiesis and digestion. It is useful as a reagent for the diagnosis of diseases and conditions including, but not limited to ductal disorders and interstitial adenomyomatosis. Similarly, polypeptides and antibodies directed against these polypeptides include
It is useful for supplying immunological probes for differential identification of tissue (s) or cell type (s). For some disorders of the tissues or cells mentioned above, especially for the immune and digestive system, certain tissue or cell types (eg hematopoiesis, immunity, reproduction, digestive organs, endocrine and cancerous and wound tissues) or body fluids (eg. : Lymph, serum,
Plasma, urine, synovial fluid and spinal fluid) or other tissue or cell samples taken from individuals with such disorders, standard gene expression levels (ie expression levels in normal tissues or fluids from individuals without the disorder) Significantly higher or lower expression of the protein as compared to can be routinely detected.

Bone marrow cells combined with the detected calcium flux and EGR1 bioactivity,
Tissue distribution in fetal liver and kidney shows that the polynucleotides and polypeptides corresponding to this gene are useful in immune and gastrointestinal disorders, and interstitial adenomyomatosis, especially tumor and proliferative disorders. More specifically, since stromal cells are important for the production of hematopoietic lineage cells, polynucleotides and polypeptides corresponding to this gene can be used in anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia, etc. It is useful in the treatment and diagnosis of disorders associated with hematopoiesis. The polypeptides and polynucleotides of the present invention are incorporated by reference in their entirety, WO
It can be used to enhance hematopoiesis as described in 9831385. Applications include bone marrow cell ex vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy for neoplasia (tumor). The gene product is also involved in lymphopoiesis,
It can also be used for immune disorders such as infection, inflammation, allergies, and immunodeficiency. In addition, the gene products may have commercial utility in the proliferation of stem cells, precursors of diverse blood lineages, and the differentiation and / or proliferation of diverse cell types. The protein, as well as the antibodies directed against the protein, may show utility as tumor markers and / or immunotherapeutic targets for the tissues listed above. In addition, the gene product of 181-16-2-0-A7-CS was shown to activate the EGR1 promoter element, which probably activates the EGR1 signaling activity in fibroblasts. Recent data indicate that EGR1 activation is involved in wound repair. An early growth response factor (Egr1), a cellular transcription factor, is expressed immediately after acute injury and serves to stimulate the production of a class of growth factors that play a role in promoting tissue repair. Egr1 expression at the rodent dermal wound site promotes angiogenesis, increases collagen production, and promotes wound closure in vitro and in vivo.
These results indicate that Egr1 gene therapy promotes the normal healing process (
Human Gene Ther 2000, vol 11 (15): 2143-58). Therefore, activators of EGR1 signaling, in particular the gene products (polypeptides and polynucleotides) of 181-16-2-0-A7-CS are incorporated by reference in their entirety in WO9941282 and WO993.
Useful in the wound healing process using the method described in 2135. <Protein of SEQ ID NO: 305 (Internal Designation No. 187-37-0-0-c10-CS)> The protein of SEQ ID NO: 305 encoded by the cDNA of SEQ ID NO: 64 is highly expressed in the prostate and brain. The protein of the present invention is human (GNP accession numbers: U95006 and U95007) and mouse (GNP accession numbers: U95003, U95004, and U95005).
) Has high homology with the D9 protein found in both. D9 is a myelocytic precursor protein transcript regulated by retinoic acid receptor α, referred to below as RAR-α (Scott et al. Blood 1996; 88: 2517-30).

Retinoic acid is an active metabolite of vitamin A and contributes to a wide range of biological processes such as cell differentiation, embryogenesis, and tumor suppression. More specifically, retinoic acid stimulates the differentiation of myeloid precursors into mature granulocytes. For example, in vitro treatment of acute promyelocytic leukemia blasts with retinoic acid induces its differentiation (Mi
Yauchi et al., Leuk Lymphoma 1999; 33: 267-80). Furthermore, treatment with retinoic acid can induce disease remission in patients with promyelocytic leukemia by causing differentiation of granulocyte precursors (Slack et al. Ann Hematol 2000; 79: 227-38).
. A diverse range of responses to retinoic acid are mediated by three receptor subtypes: RAR-α, RAR-β and RAR-γ. RAR-α has been identified as important for myeloid maturation of granulocytes (Tsai et al., Genes Dev 1992; 6: 2258-69).
Furthermore, RAR-α is arguably implicated in acute promyelocytic leukemia cells by a reciprocal translocation between the long arms of chromosomes 15 and 17 (Alcalay et al., Proc Natl Acad.
Sci USA 1991; 88: 1977-81). This type of leukemia is the predominance of malignant promyelocytes,
Primarily characterized by severe hemorrhagic signs due to activation of the coagulation cascade and fibrinolytic system (Tallman et al., Semin Thromb Hemost. 1999; 25: 209-15).
. Chromosomal reciprocal translocations lead to the production of fusion proteins that suppress the differentiation of myeloid progenitor cells and promote their survival (Grignani et al., Cell 1993; 74, 423-431). RAR-
Transient transfection of a vector containing α into a promyelocytic cell line
It upregulates the early behavior of several genes, including D9, which is highly similar to the protein of SEQ ID NO: 305 (Scott et al. Blood 1996; 88: 2517-30). Therefore, the protein of SEQ ID NO: 305 is considered to be a myeloid-associated protein whose expression is induced by activation of retinoic acid receptors including RAR-α. In a preferred embodiment, the protein of the invention or a portion thereof is present in a biological sample.
It can be used to assay the activity of RAR-α protein or retinoic acid.
In particular, since the expression of the protein of the present invention is considered to be under direct control of the retinoic acid receptor, the level of the protein of the present invention, or the mRNA encoding the protein, is determined by the effect of retinoic acid on cells. Useful as an immediate marker with high sensitivity to. The ability to detect retinoic acid receptor activation in cells with this protein has numerous uses. For example, the proteins of the invention or portions thereof can be used to monitor the effects of retinoic acid on cells of patients undergoing retinoic acid treatment for promyelocytic leukemia (Slack et al. Ann.
Hematol 2000; 79: 227-38). Because retinoic acid treatment is associated with delayed type dose-dependent side effects, a protein-based assay of SEQ ID NO: 305 has been used in patients with promyelocytic leukemia to predict and avoid such adverse side effects. It is believed that it can be used to adjust the dose of retinoic acid administered (Slack
Et al, Ann Hematol 2000; 79: 227-38).

In other embodiments, the polypeptides and polynucleotides can be used to identify myeloid progenitors, as well as brain and prostate tissue. Myeloid progenitor cells,
And the ability to specifically visualize brain and prostate tissue (and cells derived from said tissue) has been identified, for example, for determining the origin or identity of cancer cells, and for, for example, histological slide evaluation. It is useful for several applications, including facilitating the identification of cells and tissues of. Moreover, such assays can be used to determine the extent of differentiation in myeloid progenitor cells. The invention further relates to in vitro assays and diagnostic kits based on the proteins of the invention or parts thereof. Such assays can be used to diagnose disorders in which the activity of the protein is abnormally downregulated, such as blood disorders including cancer and leukemia. Since the protein of SEQ ID NO: 305, RAR- (, and acute promyelocytic leukemia are all related, changes in the measured levels of the protein of the invention, or a portion thereof, can be detected, for example, in biological samples such as serum or plasma. Can be used as a diagnostic or screening test for acute promyelocytic leukemia, and an assay capable of detecting an abnormal level of the protein of the present invention or a part thereof can detect residual disease in acute promyelocytic leukemia. Such assays can be used to assist in making therapeutic decisions in this disorder, such as, for example, whether or not more aggressive treatment is to be performed, duration of treatment, etc. In other embodiments, a variety of Various methods modulate activity and / or expression of the protein of SEQ ID NO: 305 for the treatment, attenuation and / or prevention of disorders. Can be used for. In one embodiment, for example, SEQ ID NO: 305
SEQ ID NO: a polynucleotide encoding the protein or fragments thereof of
305 protein itself, or any of a number of reagents such as compounds that increase the expression or activity of the protein of SEQ ID NO: 305, can be used in a variety of diseases, including blood disorders such as cancer, leukemia and neutropenia. It can be administered to patients suffering from or at risk of developing the disorder. For example, but not limited to, SEQ ID NO:
A protein or other compound capable of enhancing the expression or activity of the protein of 305 is administered to a patient suffering from an acute promyelocytic leukemia in order to induce the differentiation of the cells into mature granulocytes. Can be administered (Slack et al., Ann Hematol 2000
79: 227-38). In yet another preferred embodiment, the protein or other compound capable of increasing the expression or activity of a protein of the invention comprises neutropenia to induce in vivo differentiation of myeloid precursors into mature granulocytes. Alternatively, it can be used to treat, prevent, and / or attenuate agranulocytosis patients. In yet another preferred embodiment, proteins or other compounds capable of increasing the expression or activity of the protein of SEQ ID NO: 305 can be used to treat coagulation disorders such as thrombosis or hemorrhagic symptoms. For example, they can be used to treat the disseminated intravascular coagulation, a severe hemorrhagic syndrome. In this embodiment, acute promyelocytic leukemia is frequently associated with disseminated intravascular blood coagulation (Tallman et al., Semin Thromb Hem
ost 1999; 25: 209-15), supported by the fact that disseminated intravascular coagulation is effectively corrected by retinoic acid (Dombret et al., Leukemia 1993; 7: 2-9).

Further, modulation of the expression or activity of the protein of the present invention (modulation) can be used, for example, to modulate the differentiation of promyelocytic leukemia cells. In one such embodiment, the proteins of the invention maintain the undifferentiated state of cells grown in vitro using, for example, antisense molecules, antibodies, or small molecule inhibitors of expression or activity of the proteins. To be suppressed. Alternatively, agents that increase expression or activity of the protein in cells can be used to induce cell differentiation, eg, in preparing a particular cell type in vitro for a particular therapeutic application. <Protein of SEQ ID NO: 248 (Internal designation number 105-035-2-0-C6-CS) and SEQ ID NO: 313 (Internal designation number 188-28-4-0-D4-CS)> Coded by cDNA of SEQ ID NO: 7 The protein of SEQ ID NO: 248 and the protein of SEQ ID NO: 313 encoded by the cDNA of SEQ ID NO: 72 are highly expressed in brain, liver, pancreas and testis. The protein of the present invention is a nuclear protein (
Miller et al., J Biol Chem 2000; 275: 32052-6), presenting a transmembrane segment from amino acids 58-78. These proteins are homologous to human RNA polymerase II elongation factor ELL3 (EMBL Accession No. AF276512; Miller et al., J Biol Chem. 2000).
275: 32052-6). In addition, the proteins of SEQ ID NO: 248 and SEQ ID NO: 313 are
It shares sequence homology with two other members of the polymerase II elongation factor family, ELL and ELL2. The protein of SEQ ID NO: 313 is similar to the N-terminal sequence of the protein of SEQ ID NO: 248, but differs after 240 residues due to a frameshift resulting in a premature stop in the sequence of SEQ ID NO: 72 ( Miller et al., J Biol Che
m 2000; 275: 32052-6). In addition, the protein of SEQ ID NO: 248 and occuldin, an integral membrane protein found in tight junctions (Furuse et al., JC
ell Biol 1994; 127: 1617-26)
It was revealed to have 26% homology over a segment of 8 amino acids, displaying a C-terminal ZO-1 binding domain. The protein of SEQ ID NO: 313 is its C
This domain lacks because the terminal region is divergent compared to the protein of SEQ ID NO: 248. ZO-1 is part of a family of membrane-associated guanylate kinase homologs (MAGUK) that are believed to be important for signal transduction originating from cell-cell contact sites (Willott et al., Proc Natl Acad Sci USA. 1993; 90: 7834-8).

The proteins of SEQ ID NOs: 248 and 313 are RNA polymerase II elongation factors that increase the catalytic rate of transcription elongation, the phase in which RNA polymerase II migrates along DNA and extends the growing RNA chain. (Miller et al., J Biol Chem 2000; 275: 32.
052-6). In particular, the proteins of SEQ ID NOs: 248 and 313 suppress transient transcriptional attenuation at multiple sites along the DNA, thereby causing the Km and / or Vm of the polymerase to be reduced.
Modify ax (Miller et al., J Biol Chem 2000; 275: 32052-6). This protein contains one viral encoded protein (Tat) and six cellular proteins (
It belongs to a family known to include SIX, P-TEFb, TFIIF, Elongin (SIII), ELL and ELL2). Increasing empirical evidence from studies suggests that misregulation of elongation may be a key element in various human diseases (Aso et al., J Clin Invest 1996; 97: 156).
See 1-9). For example, two RNA polymerase II elongation proteins are oncogenic ELLs (Thirman et al., Proc Natl Acad) that are frequent targets of translocations in acute myelocytic leukemia.
Sci USA 1994; 91: 12110-4; Mitani et al., Blood 1995; 85: 2017-24), and controlled by the product of the von Hippel-Lindau tumor suppressor gene, which itself is associated with most clear cell kidney cancer and von. Is associated with elongin, a transcription factor that is mutated in Hippel-Lindau disease (Duan et al., Scienc
e 1995; 269: 1402-6, Kibel et al., Science 1995; 269: 1444-6). In addition, ELL
Overexpression leads to fibroblast transformation (Kanda et al., J Biol Chem. 1998 27).
273: 5248-52). Thus, the proteins of SEQ ID NOs: 248 and 313 may be important in the carcinogenesis of multiple types of neoplastic diseases, especially hematological malignancies. In one embodiment, the protein is used to increase in vitro transcription rate. Such increases can be used, for example, in any of the numerous in vitro transcription reactions routinely used for RNA preparation, protein production, characterization of promoters and transcription factors, and the like. In another embodiment, the invention provides a diagnostic tool for detecting mutations in the gene encoding SEQ ID NO: 248 or 313. Such mutations are SSCP,
Or RNA degrading enzyme and S1 protection assay, with or without denaturing factors such as DGGE, electrophoretic mobility modification of in-gel DNA fragments; dHPLC; and DNA
It can be detected by a variety of techniques, including direct sequencing. Detection of mutations in the gene encoding SEQ ID NO: 248 or 313 is useful in the detection of some diseases and conditions, such as hematological malignancies including cancer and leukemia. For example, the RNA polymerase II elongation factor ELL gene frequently translocates in acute myelocytic leukemia (Thirman et al., Proc Natl Acad Sci USA 1994; 91: 12110-4; Mitani et al., Bl.
ood 1995; 85: 2017-24), and possibly other elongation factors involved in other such diseases.

Another embodiment of the invention uses the protein or part thereof for the specific visualization of myeloid progenitor cells and pancreatic, liver, and testis tissues (and cells derived from said tissues), Compositions and methods. The ability to detect such cell types includes, for example, determining the origin and identity of cancerous cells, and facilitating identification of specific cells and tissues, for example for evaluation of histological slides. , Useful for numerous applications. Moreover, such methods can be used to investigate the degree of differentiation in myeloid or myeloid progenitor cells for staging leukemia or other neoplastic disorders. Any method for detecting the protein of the invention, or a nucleic acid encoding the protein, can be used, including methods involving the use of antibodies immunospecific for the protein of the invention. Such antibodies can be used in a variety of methods including radioimmunoassays, competitive-binding assays, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), or other techniques known to those of skill in the art. In other embodiments, the proteins of the invention, or portions thereof, can be used in the treatment, attenuation, and / or prevention of conditions associated with an unbalanced amount and / or activity of the protein of SEQ ID NO: 248 or 313. Other modulators including compounds such as agonists and antagonists, antisense and ribozyme sequences, and compounds such as antibodies can also be used in such embodiments. In a preferred embodiment, such substances are used in the treatment or prevention of certain types of neoplastic disorders associated with hematological malignancies such as cancer or leukemia. In an embodiment in which the increased expression or activity level of the protein correlates with the presence of a disease such as cancer, an antibody, an antisense molecule, a ribozyme, a dominant negative form of the protein, a compound that suppresses the expression or activity of the protein, etc. , An agent capable of causing a decrease in the activity or expression level of the protein can be used to treat or prevent the disease. Alternatively, when a decrease in the expression or activity level of the protein correlates with the presence of a disease such as cancer, a polynucleotide encoding the protein, a purified form of the protein, or an increase in the expression or activity of the present invention is used. Agents that can cause an increase in the expression or activity of the protein, such as the resulting compound, can be used to treat the disease. Furthermore, detection of the correlation between the expression or activity level of the protein and the presence or absence of the disease can be used to develop the disease itself or a diagnostic or screening tool for the detection of the predisposition to the disease.

Uses of Antibodies Antibodies of the invention include, but are not limited to, methods known in the art for purifying, detecting, and targeting polypeptides of the invention, including in vitro and in vivo diagnostic and therapeutic methods. Not have a use. Examples of such applications using immunoaffinity chromatography are listed below. The antibody of the present invention can be used alone or in combination with other compositions. For example, antibodies have use in immunoassays for qualitatively and quantitatively measuring the levels of antigenic substances in a biological sample, including a polypeptide of the invention (see, eg Harlow et al., 1988. reference). (The entire text is incorporated as a reference). Antibodies can also be used in therapeutic compositions to kill cells expressing the protein or to reduce levels of the protein in the body. The invention further relates to antibodies that act as agonists or antagonists of the polypeptides of the invention. For example, the present invention includes antibodies that partially or completely disrupt receptor / ligand interactions with the polypeptides of the present invention. Both receptor-specific and ligand-specific antibodies are included. Receptor-specific antibodies are included that do not interfere with ligand binding, but prevent receptor activation. Receptor activation (ie, signal transduction) can be determined by techniques otherwise described herein and known in the art. Also included are receptor-specific antibodies that prevent both ligand binding and receptor activation. Similarly, a neutralizing antibody that binds a ligand and prevents binding of the ligand to the receptor, as well as a ligand that binds and thereby prevents receptor activation, but the ligand does not bind to the receptor Antibodies that do not prevent are included. Antibodies that activate the receptor are also included. These antibodies may act as agonists of all or less than all biological activities affected by ligand-mediated receptor activation. Antibodies can be specified as agonists or antagonists for biological activity, including certain activities disclosed herein. The above antibody agonist can be produced using a method known in the art. For example, WO 96/40281; US Pat. No. 5,811,097; Deng et al., (1998).
Chen et al., (1998); Harrop et al., (1998); Zhu et al., (1998); Yoon et al.
(1998); Prat et al., (1998); Pitard et al., (1997); Liautard et al., (1997)
Carlson et al. (1997); Taryman et al. (1995); Muller et al. (1998):
See Bartunek et al., (1996) (said reference is incorporated by reference in its entirety).

As discussed above, the antibodies of the polypeptides of the invention may then be used to produce anti-idiotypic antibodies that “mimic” the polypeptides of the invention using techniques known to those of skill in the art. (Eg Greenspan and Bona (1989) and Nissin
off (1991), the disclosure of which is incorporated herein by reference in its entirety). For example, an antibody that binds and competitively inhibits polypeptide multimerization or binding of a polypeptide of the invention to a ligand may "mimic" a polypeptide multimerization or binding domain, resulting in , Can be used to produce anti-idiotypes that bind and neutralize the polypeptide or its ligand. Such neutralizing anti-idiotypic antibodies can be used to bind a polypeptide of the invention or its ligand / receptor and thereby block biological activity. Immunoaffinity Chromatography Antibodies prepared as described herein are attached to a support. The antibody is preferably a monoclonal antibody, but a polyclonal antibody can also be used. Supports are Sepharose CL-4B (Pharmacia, Piscataway, NJ), Sepharose CL
-2B (Pharmacia, Piscataway, NJ), Affi-gel 10 (Biorad, Richmond, C
It may be any of those typically used in immunoaffinity chromatography, including A) or glass beads. The antibody can be bound to the support using any of the coupling reagents typically used in immunoaffinity chromatography, including cyanogen bromide. After binding the antibody to the support, the support is contacted with a sample containing the target polypeptide for which isolation, purification, or enrichment is desired. This target polypeptide is SEQ ID NO: 242.
~ 482, the mature polypeptides contained in SEQ ID NOS: 242-272 and SEQ ID NOS: 274-384, as well as the full-length or mature polypeptides encoded by the clone inserts of the deposited clone pool, variants and fragments thereof, or the selection polymorphs described above. It can be a polypeptide selected from the group consisting of fusion proteins comprising peptides or fragments thereof. Preferably, the sample is placed in contact with the support for a sufficient period of time and under suitable conditions to allow at least 50% of the target polypeptide to specifically bind to the antibody bound to the support. To do.

The support is then washed with a suitable wash solution to remove the polypeptide that is not specifically attached to the support. This washing solution is PBS, Tris-lithium chloride buffer (0.1M lysine base 0.5M lithium chloride, pH 8.0), Tris-hydrochloric acid buffer (
0.05M Tris hydrochloride, pH 8.0) or Tris / Triton / sodium chloride buffer (50mM hydrochloric acid, pH 8.0 or 9.0, 0.1% Triton X-100, and 0.5M
Any of those typically used in immunoaffinity chromatography, including sodium chloride). After washing, the specifically bound target polypeptide is eluted from the support using high pH or low pH eluents typically used in immunoaffinity chromatography. In particular, the eluent may include an eluent such as triethanolamine, diethylamine, calcium chloride, sodium tricyanate, potassium bromide, acetic acid, or glycine. In certain embodiments, the eluent may also include a detergent such as Triton X-100 or octyl-beta-D-glucoside. Transfer Vector The GENSET polypeptides of the present invention can also be used as carriers for transferring the so-called "shipments" of a protein or peptide of interest into tissue culture cells or host organisms. A hydrophobic region of a GENSET polypeptide or fragment thereof, preferably a signal peptide, which is a sequence selected from the group consisting of SEQ ID NOs: 1-31 and 33-143, and clone inserts of the deposited clone pool, more preferably a short signal peptide. The core hydrophobic region (h) can be used as a carrier. If a cell-penetrating peptide of limited size (approximately 25 amino acids or less) translocates across the cell membrane, the C of the cargo peptide of interest is
Chemical synthesis can be used to add h regions to either the termini or the N-terminus.
Alternatively, if a longer peptide or protein is introduced into the cell, in order to link the cDNA sequence encoding the hydrophobic region or a fragment thereof to the 5'or 3'end of the DNA sequence encoding the cargo polypeptide, Genetic engineering manipulations can be performed on nucleic acids using techniques known to those of skill in the art. Such genetically engineered nucleic acid is then translated in vitro or in vivo after transfection into suitable cells, using conventional techniques so as to produce a cell-penetrating polypeptide. Then, simply by adding the cell-permeable polypeptide to a suitable host cell and incubating, the polypeptide is translocated across the membrane.

This method can be applied to the study of diverse intracellular functions and processes. For example, it has been used to probe functionally relevant domains of intracellular proteins and to investigate protein-protein interactions involved in signaling pathways (Lin et al., J. Biol. Chem., 270: 14225-14258 (1995); Lin et al.
J. Biol. Chem., 271: 5305-5308 (1996); Rojas et al., J. Biol. Chem., 271.
: 27456-27461 (1996); Rojas et al., Nature Biotech., 16: 370-375 (1998);
Liu et al., Proc. Natl. Acad. Sci. USA, 93: 11819-11824 (1996); Rojas et al.
Bioch. Biophys. Res. Commun., 234: 675-680 (1997); Du et al., J. Peptide.
Res., 51: 235-243 (1998)). Such techniques can be used in cell therapy to transfer proteins that produce a therapeutic effect. For example, cells isolated from a patient can be treated with the transferred therapeutic protein and reintroduced into the host organism. Alternatively, the hydrophobic region of the signal peptides of the invention can be used in combination with a nuclear localization signal to deliver nucleic acids into the cell nucleus. Such oligonucleotides can be antisense oligonucleotides or oligonucleotides designed to form triple helices, as described herein, to suppress processing or maturation of target cell RNA, respectively. . (Expression of GENSET product) Spatial expression of GENSET gene of the present invention The tissue expression of the cDNA of the present invention was examined. Table IX lists Genset libraries of tissues and cell types tested to express the polynucleotides of the invention. Tissues and cells tested for expression of polynucleotides include adrenal gland (AG), bone marrow (BM
), Cerebrum (Br), cancerous prostate (CP), cerebellum (Ce), colon (Co), dystrophic muscle tissue (DM), fetal cerebrum (FB), fetal kidney (FK), fetal liver (FL) , Heart (He), hypertrophic prostate (HP), kidney (Ki), liver (Li), lung (Lu), lung cell (LC), lymph ganglion (LG), lymphocyte (Ly), muscle (Mu) ), Ovary (Ov), pancreas (Pa), pituitary gland (
PG), placenta (Pl), prostate (Pr), salivary gland (SG), spinal cord (SC), spleen (Sp), stomach / gut (SI), substantia nigra (SN), testis (Te), thyroid (Ty). , Umbilical cord (UC) and uterus (
Ut).

In each cDNA referenced by its SEQ ID NO (first column), the number of proprietary 5'ESTs (ie, cDNA fragments) expressed in the particular tissue referenced by its name is indicated after the semicolon. (Second column). Furthermore, the bias in the spatial distribution of the polynucleotide sequences according to the invention was tested by comparing the relative proportions of biological polynucleotides in a given tissue using the following statistical analysis. Underexpression or overexpression of a given cluster of polynucleotides in a given tissue was performed using the normal approximation of the binomial distribution. A chi-square test reported that a bias in frequency was "favorable" if the observed proportion of polynucleotides in a given tissue at a given consensus was less than 1% random. Table X shows the results as follows. Each polynucleotide showing a bias in tissue distribution is referenced by its SEQ ID NO: in the first column, the second column is referred to as "low frequency expression" a list of tissues in which the polynucleotide is underexpressed, A list of tissues in which the polynucleotide is overexpressed is shown in the third column entitled "High Frequency Expression." In addition, a part of the cell localization position of the polypeptide of the present invention is “psort software” (Na
kai and Horton, (1999); Nakai and Kanehisa, (1992), the disclosures of which are incorporated by reference in their entireties). The localized position below the predicted cell level of each polypeptide identified by its SEQ ID NO: in the first column is listed in the second column of Table XI. Evaluation of expression level and expression pattern of GENSET mRNA Furthermore, the spatial and temporal expression pattern of GENSET mRNA, and their expression levels can be determined as follows. The expression level and expression pattern of GENSET mRNA can be analyzed by solution hybridization using an extension probe as described in International Patent Application WO 97/05277, the description of which is fully incorporated herein by reference. is doing. Briefly, it corresponds to the gene encoding the mRNA to be characterized.
The GENSET polynucleotide, or a fragment thereof, binds to bacteriophage (T3
, T7 or SP6) Insert into the cloning site directly downstream of the RNA polymerase promoter to produce antisense RNA. Preferably, the length of the GENSET polynucleotide is a minimum of 100 nucleotides. The plasmid is linearized and transcribed in the presence of ribonucleotides containing modified ribonucleotides (eg biotin-UTP and DIG-UTP). This double-labeled RNA is isolated from cells or tissues of interest.
Hybridized with RNA in water. Hybridization is carried out under standard stringent conditions (40-50 ° C for 16 hours, formamide 80%, NaCl 0.4 M buffer, pH 7-8). The unhybridized probe is deleted by the single-stranded RNA-specific ribonuclease (ie RNase CL3, T1, Phy M, U2 or A). The presence of the biotin-UTP modification allows the hybrid to be captured on microtitration plates coated with streptavidin. The presence of the DIG modification allows the hybrid to be quantified and detected by an ELISA method using an anti-DIG antibody conjugated to alkaline phosphatase.

The GENSET cDNA, or fragments thereof, contains a serial analysis of gene expression as disclosed in British Patent Application No. 2 305 241 A.
on: SAGE), a tag can be added with a nucleotide sequence, the entire contents of which are incorporated herein by reference. In this method, cDNA is
It is prepared from a tissue, organism or other source of nucleic acid for which it is desired to determine gene expression patterns. The resulting cDNA is separated into two pools. The cDNA of each pool is cleaved by a first restriction endonuclease called an "anchoring enzyme" that has at least one recognition site present in most cDNAs. Cleaved cDNA
The fragment containing the region closest to the 5'or 3'of is isolated by binding to a capture medium such as beads coated with streptavidin. A first oligonucleotide linker having a first sequence for hybridizing an amplification primer and an internal restriction site for a "tagging endonuclease" is ligated to the first pool of digested cDNA. Digestion with a second endonuclease produces short "tag" fragments from cDNAs. A second oligonucleotide linker with a second sequence and internal restriction sites for hybridizing the amplification primer is ligated to the second pool of digested cDNA. The second pool cDNA fragment is also the second pool cDNA
Digested with "tagging endonuclease" to yield a short "tag" fragment derived from The "tags" obtained from the digestion of the first and second pools with the anchoring enzyme and the tagging endonuclease are ligated together to give a "ditag". In some embodiments, the ditag is concatamered to obtain a ligation product containing 2-200 ditags. Next, determine the sequence of tags and use GENSET c
By comparing with the sequence of DNA, it is determined which gene is expressed in the cell, tissue, organism or other nucleic acid source from which the tag was derived. In this way, the expression pattern of the GENSET gene in cells, tissues, organisms or other nucleic acid sources is obtained. Quantitative analysis of GENSET gene expression can also be performed by using an array. For example, quantitative analysis of gene expression can be performed in complementary DNA microarrays with GENSET polynucleotides, or fragments thereof, as described by Schena et al. (1995 and 1996), the disclosure of which is incorporated herein by reference in its entirety. Incorporated as a reference. The GENSET cDNAs or their fragments are amplified by PCR and placed on a chill silylated microscope slide in an array in a 96-well microtiter plate by a high speed robot. The printed array is left in a humid chamber to rehydrate the array elements and rinsed once with SDS 0.2% for 1 minute, twice in water for 1 minute, and once with sodium borohydride solution for 5 minutes. Arrays are placed in 95 ° C water for 2 minutes, 0.2%
Transfer to SDS, place for 1 minute, rinse twice with water, air dry and store at 25 ° C in dark. Cell or tissue mRNA is isolated or obtained commercially and the probe is prepared by one round of reverse transcription. The probes are hybridized to a 1 cm2 microarray under a 14 x 14 mm glass coverslip for 6-12 hours at 60 ° C. Array is 25 ° for 5 minutes
Wash with low stringency wash buffer (1X SSC / 0.2% SDS) at C, then with high stringency wash buffer (0.1X SSC / 0.2% SDS) for 10 minutes at room temperature. Arrays were scanned in 0.1X SSC using a fluorescence laser scanner equipped with a custom filter set. Accurate differential expression measurements are obtained by averaging the ratios of two independent hybridizations.

Quantitative analysis of gene expression can also be performed using GENSET cDNA or its fragments in a complementary DNA array as described by Pietu et al. (1996), the publication of which is incorporated herein by reference in its entirety. Transferred. The inventive GENSET polynucleotides or fragments thereof are PCR amplified and found on the membrane. Next, mRNA derived from various tissues or cells is labeled with a radioactive nucleotide. Hybridized mRNAs after hybridization and washing under controlled conditions
Is detected by phosphoimaging or autoradiography. Duplicate experiments are performed, followed by quantitative analysis of differentially expressed mRNAs. Alternatively, expression analysis of the GENSET gene can be performed by using Lockha
This can be done as described by rt et al. (1996) and Sosnowski et al. (1997), the disclosure of which is incorporated by reference in its entirety. Oligonucleotides of 15 to 50 nucleotides corresponding to the sequence of the GENSET polynucleotide or a fragment thereof may be synthesized directly on the chip (Lockhart et al., Supra) or after synthesis and addressed on the chip (Sosnowski et al., (See above). Preferably, the oligonucleotide length is about 20 nucleotides. The cDNA probe is labeled with an appropriate compound such as biotin, digoxigenin or a fluorescent dye, synthesized from an appropriate mRNA population and randomly fragmented to an average size of 50 to 100 nucleotides. The probe is hybridized to the chip. After washing and applying various electric fields (Sosnowsky et al., Supra) as described by Lockhart et al. (Supra), the dye or labeled compound is detected and quantified. Perform double hybridization. Comparative analysis of signal intensities from different cDNA samples and from cDNA probes on similar target oligonucleotides shows differential expression of GENSET mRNA. Uses of GENSET Expression Data Using any of the techniques known to those of skill in the art, particularly those described in the section entitled "Assessing Expression Levels and Patterns of GENSET mRNA", or the levels of GENSET mRNA expression by use of the present disclosure. And, once the pattern is determined, these information is used to design a GENSET-specific marker for the purpose of detection, identification, screening and diagnosis, or a DNA construct having an expression pattern similar to the GENSET expression pattern. Can be designed.

Detection of GENSET Expression and / or Biological Activity The present invention also uses the polynucleotide and polypeptide sequences described herein to detect GENSET expression and / or biological activity in a biological sample. Related to the method of. Such methods may be used, for example, for normal or abnormal GENSET expression and / or
Alternatively, it can be used for screening biological activity, and thus can be used diagnostically. Biological samples used in the method of the invention include suitable samples, eg of mammals, especially humans. For example, the sample may be sourced from a tissue or cell line of the same origin as the tissue or cell line in which the polypeptide is known to be expressed using the data in Table IX. Detection of GENSET Products The invention also relates to methods for detecting GENSET polynucleotides or polypeptides in a sample using the sequences described herein and any technique known to those of skill in the art. For example, a method of detecting GENSET polynucleotides in a sample can use a labeled polynucleotide probe having all or a functional portion of the nucleotide sequence of the GENSET polynucleotide. In one embodiment, the sample is treated so that the polynucleotides in the sample are available for hybridization with a polynucleotide probe, which can be DNA or RNA. The resulting treated sample is combined with a labeled polynucleotide probe having all or part of a GENSET cDNA or genomic sequence under conditions suitable for hybridization of complementary sequences. The detection of polynucleotide hybridization from a sample by a labeled nuclear probe is
Indicates that the GENSET polynucleotide is present in the sample. The presence of GENSET mRNA indicates GENSET expression. Accordingly, the present invention provides a polynucleotide comprising a nucleotide sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-241, the sequence of clone inserts of the deposited clone pool, the sequence completely complementary thereto, fragments and variants thereof. A method for detecting the presence is included. In a first embodiment, the method comprises the steps of: a) contacting the sample with a nucleic acid probe or nucleic acid probes that hybridize to the selected nucleotide sequence, and b) with the probe or probes and the poly The hybrid complex formed with the nucleotide is detected.

In a preferred embodiment of the above detection method, the nucleic acid probe or nucleic acid probes are labeled with a detectable molecule. In another preferred embodiment of the above detection method, the nucleic acid probe or the plurality of nucleic acid probes are immobilized on a substrate. In still another preferred embodiment, the nucleic acid probe or nucleic acid probes have a sequence contained in a sequence complementary to the selected sequence. In a second embodiment, the method comprises the steps of: a) contacting the sample with an amplification reaction containing two amplification primers flanking the region of the nucleotide sequence to be amplified; b) Perform an amplification reaction to synthesize an amplification product containing the region of the selected nucleotide sequence; c) detect the amplification product. In a preferred embodiment of the above detection method, the polynucleotide to be amplified is RN
If it is an A molecule, pre-reverse transcription and synthesis of a second cDNA strand are required to provide the DNA template to be amplified. In another preferred embodiment of the above detection method, the amplification product is detected by hybridization with a labeled probe having a sequence complementary to the amplified region. In yet another preferred embodiment, at least one amplification primer has a sequence contained in the selection sequence or a sequence complementary to the selection sequence. Alternatively, the method of detecting GENSET gene expression in a test sample can be performed by using any product that binds to the GENSET polypeptide or a part of the GENSET polypeptide of the present invention. Such products may be polypeptides capable of specifically binding to GENSET polypeptides or fragments thereof, including antibodies, binding fragments of antibodies, GENSET agonists and antagonists. Detection of specific binding to the antibody indicates the presence of GENSET polypeptide in the sample (eg ELISA method). Accordingly, the invention further relates to a method of specifically detecting the presence of a GENSET polypeptide according to the invention in a biological sample, which method comprises the steps of: Contacting with a product capable of binding the fragment, b) allowing the product to bind to the polypeptide and form a complex, and b) detecting the complex.

In a preferred embodiment of the above detection method, the product is an antibody. In a more preferred embodiment, the antibody is labeled with a detectable molecule. In another more preferred embodiment of the above detection method, the antibody is immobilized on a substrate. The invention also includes GENSET products (eg, polynucleotides or polypeptides).
Relates to a method of determining whether or not is present in a biological sample, the method comprising the steps of: a) obtaining the biological sample from a human or non-human animal, preferably a mammal; Contacting with a product capable of binding the GENSET polynucleotide or polypeptide of the invention, c) determining the presence or absence of the GENSET product in the biological sample. A compound that specifically binds a GENSET product is a compound that binds to a GENSET polypeptide (eg, a binding protein, antibody or binding fragment thereof (eg, F (
ab ') 2 fragment) or a compound (eg, complementary probe or primer) that binds to the GENSET polynucleotide. The present invention further relates to kits that can be used to detect GENSET expression products. This kit comprises a compound that specifically binds to a GENSET polypeptide (eg, binding protein, antibody or binding fragment thereof (eg, F (ab ′) 2 fragment), or GENSET mRNA (eg, complementary probe or primer), eg, It may be included in the container means, and the kit may further include auxiliary reagents such as a buffer solution, etc. [Detection of GENSET biological activity] Further, the present invention includes a method for specifically detecting GENSET biological activity. Assessment of GENSET biological activity can be performed using a variety of techniques, including those described in the section entitled "Erreur! Source du renvoi introuvable." A method for specifically detecting a biological activity comprising the steps of: a) obtaining a biological sample from a human or non-human mammal; Out to.

The present invention further relates to kits that can be used to detect GENSET biological activity. <Identification of a specific situation of GENSET expression> If the expression pattern of GENSET mRNA shows that the GENSET gene is specifically expressed in a predetermined situation, a probe specific to this gene and Primers, as well as antibodies that bind GENSET polynucleotides, can be used as markers for specific situations. Examples of specific situations include specific expression in a given tissue / cell or tissue type / cell type, expression at a given stage in the development of a process such as embryogenesis or disease development, or in a given organelle. Specific expression is mentioned. Such primers, probes and antibodies can be used, for example, to identify forensic samples, tissues / cells / organs of unknown origin in differentiated tumor tissues that have metastasized to foreign sites, or include, for example, in situ PCR or immunochemistry. , Commercially useful for distinguishing various tissue types in a tissue cross section by using any technique known to those skilled in the art. For example, using cDNAs and proteins in the sequence listing, and their fragments,
A distinction may be made between human tissues / cells and non-human tissues / cells, or human tissues / cells / organs may or may not express a polynucleotide containing a cDNA. By knowing the expression pattern of a given GENSET by routine experimentation or use of the present disclosure, the polynucleotides and polypeptides of the present invention can be identified in unknown tissue / cell samples /
It can be used in a method of determining the identity of organelles. As part of a method of determining the identity of an unknown tissue / cell sample / organelle, the poly of the present invention can be used to determine what is identical to an unknown tissue / cell sample and what is not. Nucleotides and polypeptides may be used. For example, if the cDNA is expressed in a particular tissue / cell type / organ and the unknown tissue / cell sample / organ does not express the cDNA, the unknown tissue / cell is not human or the cDNA is It can be presumed to be an unknown tissue / cell type / organelle different from the one expressed. A method for determining the identity of these tissues / cells / organelles is to use mRNA (or corresponding
The presence or absence of cDNA) is detected by a method known in the art (for example, hybridization, PCR-based method, immunoassay method, immunochemistry, ELISA, etc.). Below, an example of such a technique is described in more detail. Accordingly, the invention encompasses the use of the polynucleotides and polypeptides of the invention as tissue markers. In a preferred embodiment, for this purpose, preferably Table X
The polynucleotide expressed in a given tissue as shown in, and the polypeptide encoded by such a polynucleotide are used. The invention further encompasses the use of the polypeptides of the invention as organelle markers. In a preferred embodiment, the polypeptides expressed in the predetermined subcellular compartments shown in Table XI are preferably used for this purpose.

Accordingly, the present invention includes a method of distinguishing between tissue / cell type / subcellular compartments, which method comprises the steps of: a) a biological sample whose identity is to be assayed as a GENSET product. Contacting with a product capable of binding, b) determining whether the GENSET product is expressed in the biological sample. Products capable of specifically binding to a GENSET product, ie to a GENSET polypeptide or GENSET mRNA, include binding proteins, antibodies or binding fragments thereof (eg F (ab ′) 2 fragments), and GENSET complementary probes and Includes primers. Step b) is carried out using any of the detection methods known to the person skilled in the art, including those disclosed herein, especially those disclosed in the section entitled "Detection of GENSET expression and / or biological activity". be able to. [Identification of Tissue Type or Cell Type by Means of Tissue-Specific Labeled Antibody] Specific tissue identification is accomplished by antibody preparation coupled directly (eg green fluorescent protein) or indirectly detectable marker. This is achieved by visualization of the tissue-specific antigen by means of a material. The selected labeled antibody type binds to the binding partner of the specific antigen in the tissue cross-section, cell suspension or extract of the soluble protein of the tissue sample and provides a pattern for qualitative or semi-qualitative interpretation. The antiserum used in these procedures should exceed that of the preparation whose potency was unchanged, so that the antibody may be expressed in mg by isolation of the gamma globulin fraction, for example by ion exchange chromatography or ammonium sulfate fractionation. / ml concentration. Furthermore, in order to provide the most specific antisera, unwanted antibodies, such as those directed against common proteins, will be isolated from the gamma globulin fraction before the antibody is labeled with a marker, for example by means of an insoluble immunosorbent. Need to be removed. Antisera, whether monoclonal or heterologous, are suitable in any procedure. A. Immunohistochemistry Techniques High titer antibodies prepared and purified as described above are described in, for example, Fudenberg et al. (1980).
Alternatively, it is linked to a detectable marker as described by Rose et al. (1980), the disclosure of which is incorporated herein by reference in its entirety.

The antibody is preferably a fluorescent marker of fluorescein or rhodamine, but may be labeled with an enzyme such as horseradish peroxidase that corresponds to the chromogenic reaction with a substrate. It is also possible to add a marker in a second step to the antibody bound to the tissue as described below. Alternatively, the specific anti-tissue antibody can be labeled with ferritin or other electron-dense particles and the localization of the ferritin-bound antigen-antibody complex is performed using electron microscopy. In yet another alternative, the antibody can be detected, for example, by radiolabelling the antibody with 125 I and coating the antibody-treated preparation with photographic emulsion. The preparation for carrying out the procedure may comprise a monoclonal or polyclonal antibody against a single protein or peptide recognized as being specific for the tissue type, for example brain tissue, or a plurality of antigenically distinct tissue-specific antigens. The antibody preparations against can optionally be used on a panel, either individually or as a mixture. Tissue sections and cell suspensions are prepared for immunohistochemistry according to common histology techniques. Mount multiple cryostat sections of unknown tissue and known controls (approximately 4 um, non-immobilized) and cover each slide with various dilutions of the antibody preparation. Cross sections of known and unknown tissues should also be treated with the preparation to provide positive controls, negative controls, eg preimmune serum, and controls for non-specific staining, eg buffer. The treated sections are left for 30 minutes at room temperature in a humid chamber, rinsed and then washed with buffer for 30-45 minutes. Soak up excess liquid and develop the marker. If the tissue-specific antibody was not labeled in the first incubation, it can now be labeled by the second antibody-antibody reaction. This can be achieved, for example, by adding an antibody to an immunoglobulin class of antiserum producing species, eg fluorescein-labeled antibody to mouse IgG, conjugated with fluorescein or an enzyme. Such labeled serum is commercially available. The antigen detected in the tissue by the above process can be quantified by measuring the intensity of the color or fluorescence on the tissue cross section and calibrating the signal using an appropriate standard. B. Identification of tissue-specific soluble proteins Visualization of tissue-specific proteins and identification of unknown tissues in the process can be performed using labeled antibody reagents and detection strategies as described in immunohistochemistry. , Samples are prepared according to electrophoretic techniques to distribute proteins extracted from tissues into arrays arranged for detection by molecular weight. The tissue sample is homogenized using a Virtis instrument, and the cell suspension is disrupted by milling homogenization or osmotic lysis, in each case as it normally occurs in the art. Use detergent as needed for disruption. Nucleus, microsome,
Insoluble cell components such as membrane fragments and the like are removed by ultracentrifugation, and the fraction containing soluble protein is concentrated if necessary and dedicated for analysis. A sample of the soluble protein solution is separated into individual proteins by conventional SDS polyacrylamide gel electrophoresis as described, for example, in Davis et al., Section 9-2 (1986). At this time, the size markers are moved to equilibrium in order to estimate the molecular weight of the constituent proteins using several different amounts of polyacrylamide so that the entire molecular weight range of the protein to be detected in the sample can be separated. The sample size for analysis is a convenient amount of 5 to 55 ul and contains 1 to 100 ug of protein. An aliquot of each degraded protein is transferred to a nitrocellulose filter by blot on transfer paper. This process preserves the separation pattern. Prepare multiple copies. The process known as Western blot analysis is described in detail in Davis et al., (1986) Section 19-3. For comparison with antibody-bound proteins, a series of nitrocellulose blots are stained with Coomassie blue dye to visualize the entire series of proteins. The remaining nitrocellulose filter is then incubated with one or more solutions of specific antisera directed against tissue-specific proteins, prepared as described in this application. In this process, as in process A above, the appropriate positive and negative samples and reagent controls are transferred in either process A or B according to various strategies and their permutations. It can bind to the antigen-first antibody complex. In the direct method, the first specific antibody can be labeled. Alternatively, the unlabeled complex can be bound to a labeled second anti-IgG antibody. In other methods, either the first or second antibody is linked to a biotin molecule, which is capable of binding an avidin binding marker in the following steps. According to yet another strategy, enzyme-labeled or radioactive protein A, which has the property of binding to any IgG, is bound in the final step to either the first or second antibody. Visualization of tissue-specific antigens against one or more tissue-specific antibodies that have been prepared from the gene sequences identified by the cDNA sequences and that bind at levels greater than those found in control tissues, for example forensic It is possible to identify tissues of unknown origin such as various samples and differentiated tumor tissues that have metastasized to foreign bodies.

Targeting compounds to subcellular compartments Specific gene compartment / organelle expressed GENSET polypeptides should be used to target compounds to these cell compartments / organelles. Is also possible. Accordingly, the invention encompasses the use of the polynucleotides and polypeptides of the invention as organelle targeting tools. In a first embodiment, GENSET polypeptides expressed in mitochondria can be used to target heterologous compounds, either polypeptides or polynucleotides, to mitochondria. In this case, a fragment of the protein of the present invention is genetically or chemically fused to a heterologous polypeptide or polynucleotide. Preferred fragments are signal peptides, fragments of the amphipathic alpha helix and / or any other protein of the invention, or parts thereof, including target signals for mitochondria, including but not limited to matrix targeting signals, These are Herrman and Neupert, (2000),
Bhagwat et al. (1999), Murphy (1997); Glaser et al. (1998); Ciminale et al. (1999),
And their disclosures are incorporated herein by reference in their entireties. Such heterologous compounds can be used to modulate mitochondrial activity. These can be used, for example, to induce and / or prevent mitochondria-induced apoptosis or necrosis. Also,
Heterologous polynucleotides can be used in mitochondrial gene therapy to replace defective mitochondrial genes and / or to inhibit the toxic expression of certain mitochondrial genes. In a second embodiment, GENSET polypeptides expressed in the endoplasmic reticulum can be used to target heterologous polypeptides to the endoplasmic reticulum. At this time, a fragment of the protein of the present invention is genetically or chemically fused to a heterologous polypeptide.
A preferred fragment is any fragment of the protein of the invention, or a portion thereof, which may include an endoplasmic reticulum targeting signal, these are Pidoux
And Armstrong (1992), Munro and Pelham (1987); Pelham (1990), the disclosures of which are incorporated herein by reference in their entireties.

In a third embodiment, nuclear-expressed GENSET polypeptides can be used to target heterologous polypeptides or polynucleotides to the nucleus. At this time, a fragment of the protein or polynucleotide of the present invention is genetically or chemically fused to a heterologous polypeptide or polynucleotide. Preferred fragments are any fragment of the protein of the invention, or parts thereof, which may include targeting signals for the nucleus (nuclear localization signals), which are described in Christophe et al., (2000). The entire text of this publication is incorporated herein by reference. In a fourth embodiment, the nuclear expressed GENSET polypeptide can be used to target a heterologous polypeptide to the Golgi apparatus. At this time, a fragment of the protein of the present invention is genetically or chemically fused to a heterologous polypeptide. Preferred fragments include any of the signal peptides, transmembrane domains, tyrosine-containing regions and / or other fragments of proteins of the invention, or portions thereof (1) Ugur and Jones, (2000); Picetti and Borrelli,
Target signals for the Golgi apparatus as described in (2000), (2) Tyrosine-based Golgi target signal region (Zhan et al., (1998); Watson and Pessin (2000); Ward.
And Moss (2000), or (3) Munro, (1998); Luetterforst et al., (1999); E.
ssl et al., (1999), which may include any other areas, the disclosures of which are incorporated herein by reference in their entireties. <Screening and diagnosis of abnormal GENSET expression and / or biological activity> Furthermore, antibodies and / or primers specific for GENSET expression are
After recognizing expression and / or biological activity, it can also be used to screen and / or diagnose disorders associated with aberrant GENSET expression. For example,
Certain diseases may result from lack of expression, overexpression, or underexpression of GENSET mRNA. In a sample obtained from a healthy person, the expression pattern and expression amount of mRNA,
By comparing with those obtained from an individual suffering from a particular disorder, the gene responsible for this disorder can be identified. In that case, using a probe, primer and antibody specific for this GENSET, the gene of interest is specifically expressed, or its expression is specifically unregulated, that is, overexpression or underexpression. It is possible to create kits for screening and diagnosing existing disorders.

[Screening for Specific Disorders] The present invention also relates to a method for identifying an individual exhibiting increased or decreased GENSET levels, which may contribute to a therapeutic method for suppressing or amplifying GENSET expression, respectively. . An example of such a method comprises the steps of: a) obtaining a biological sample from a human or non-human mammal, b) as disclosed herein, particularly as disclosed in the section entitled "GENSET Product Detection". GE in the sample using any of the detection methods known to those of skill in the art, including
Detecting the presence of NSET product (mRNA or protein), c) comparing the amount of the GENSET product present in the sample with that of a control sample; and d) the GENSET expression level of the human or non-human mammal is a control. Determine whether it shows a decrease or increase compared to the sample. Individuals who exhibit reduced or elevated levels of such GENSET products may be predisposed to develop disorders associated with dysregulated GENSET gene expression and are therefore candidates for treatment. The identification of elevated GENSET levels in patients has been characterized by GENS
It indicates an individual who is considered to contribute to the treatment with a formulation that suppresses ET expression or activity. The identification of GENSET levels that show a decrease in patients indicates that the individual is likely to contribute to treatment with a formulation that induces GENSET expression or activity. Biological samples suitable for use in this method include biological fluids such as blood, lymph, saliva, semen, breast milk, and tissue samples (eg, biospecimens), as well as cell cultures or cell extracts derived from, for example, tissue biospecimens. including. The detection step of the method may be performed using a general protocol for protein / mRNA detection. Suitable protocols include Northern blot analysis immunoassays (eg RIA, Western blot,
Immunohistochemical analysis) and PCR. Accordingly, the present invention further relates to a method of identifying an individual or non-human animal at high risk of developing, or currently having, certain diseases / disorders associated with aberrant expression of the GENSET gene or biological activity. An example of such a method comprises the steps of: a) obtaining a biological sample from a human or non-human mammal, b) detecting the presence or absence of a GENSET product (mRNA or protein) in the biological sample, and c) the sample. Comparing the amount of the GENSET product present in a control sample; and d) the human or non-human mammal is at increased risk of developing a disease or disorder,
Alternatively, it is determined whether or not it is currently in such a state.

According to this method, the presence of altered levels of GENSET product in the sample indicates that the subject is predisposed to developing the disease / disorder. Biological samples suitable for use in this method include biological fluids such as blood, lymph, saliva, semen, breast milk, and tissue samples (eg, biopsies. Diagnostic methodologies described herein include human and non-human mammals). [Detection of GENSET Mutations] The present invention further relates to methods of detecting mutations in the GENSET polynucleotides of the present invention, such methods comprising in the GENSET gene and preferably those Can be advantageously used to detect mutations that occur in the control region of E.coli.If mutations are determined to be associated with disease, screening for such mutations can be used for screening and diagnosis. In one embodiment of the oligonucleotide array of the present invention, abrupt changes that occur in the GENSET gene and preferably in their control regions. An oligonucleotide probe matrix can be conveniently used to detect a. For this particular purpose, the probe may be a known mutation (either a deletion, insertion or substitution of one or more nucleotides). It has been specifically designed to have a nucleotide sequence that allows it to hybridize with a gene having a mutation in the GENSET gene, which is For example
Identified by the techniques used by Huang et al. (1996) or Samson et al. (1996), the disclosures of which are incorporated herein by reference in their entireties. Another technique used to detect mutations in the GENSET gene is high density
The use of DNA arrays. Each oligonucleotide probe that forms the unit element of a high-density DNA array is designed to match a specific subsequence of GENSET genomic DNA or cDNA. Therefore, using an array consisting of oligonucleotides complementary to a partial sequence of the target gene sequence, the identity between the target sequence and the natural gene sequence is determined, the amount is measured, and the target sequence of the GENSET gene is compared with the reference natural sequence. Detect differences between gene sequences. One such design, which is named 4L tile array, has a set of 4 probes (A, C, G, T), preferably 15 probes.
An oligomer of nucleotides has been introduced. In each set of 4 probes, the perfect complement hybridizes more strongly than the mismatched probe. As a result, a length L nucleic acid target was scanned for mutations with a 4L probe containing a tile array, and the entire probe set contains all possible mutations in a known natural reference sequence. The hybridization signal from the tile array of 15-mer probe sets is perturbed by a single base change in the target sequence. As a result, probes on either side of the mutation site exhibit a characteristic signal loss or "footprint". This method was described by Chee et al. In 1996, the disclosure of which is incorporated herein by reference in its entirety.

<Preparation of DNA Construct Having GENSET Expression Pattern> Furthermore, as described below, the spatial and temporal expression pattern of GENSET mRNA and the characterization of the expression level are also as described below. It is useful to create expression vectors that can produce the desired level of gene product in the desired spatial or temporal form. DNA constructs capable of directing temporal and spatial GENSET gene expression in recombinant cell hosts and transgenic animals: Physiologic lack of synthesis of GENSET proteins at both cellular and multicellular organism levels To examine phenotypic consequences, the present invention also provides DNA constructs and recombinant vectors that enable conditional expression of GENSET genomic sequences or cDNAs, and specific alleles of copies of the genomic sequences or cDNAs. Including and copying is
SEQ ID NOs: 1-241 and the sequence of the clone insert of the deposited clone pool, or with a nucleotide sequence selected from the group consisting of fragments thereof, having one or more base substitutions, deletions, or additions. The substitutions, deletions or additions are located within exons, introns or regulatory sequences, but are preferably within the 5'regulatory sequences or exons of the GENSET genomic sequence or within the GENSET cDNA. The first preferred DNA construct is one that regulates GENSET gene expression based on the tetracycline resistance operon tet of E. coli transposon Tn10, such as Gos
sen et al. (1992, 1995) and Furth et al. (1994), the disclosure of which is incorporated herein by reference in its entirety. Such a DNA construct is T
It contains seven tet operator sequences (tetop) from n10, which are fused to either the minimal promoter or the 5'regulatory sequence of the GENSET gene, wherein the minimal promoter or the GENSET regulatory sequence is a sense or antisense oligonucleotide. Alternatively, it is operably linked to a polynucleotide of interest encoding a polypeptide, including a GENSET polypeptide or a peptide fragment thereof. this
The DNA construct was prepared in the same cell with additional HCMV IE1 enhancer / promoter or MM
If it contains a nucleotide sequence encoding a native (tTA) or mutant (rTA) repressor fused to the activation region of the herpes simplex virus viral protein VP16 under the control of a promoter such as TV-LTR, then Is functional as a conditional expression system for the nucleotide sequence of. Indeed, preferred DNA constructs of the invention are those that contain both a polynucleotide with a tet operator sequence and a polynucleotide with a sequence encoding the tTA or rTA repressor. In a specific embodiment, the conditional expression DNA construct comprises a rTA-encoding sequence that is a repressor of a mutant tetracycline, and expression of the polynucleotide of interest is silent in the absence of tetracycline and induced in its presence. To be done.

DNA Constructs Allowing Homologous Recombination: Replacement Vectors A second preferred DNA construct contains, from the 5 ′ end to the 3 ′ end, the following:
(a) The first nucleotide sequence contained in the GENSET genome sequence; (b) Neomycin resistance (
a nucleotide sequence containing a positive selection marker such as the neo) marker; and (c
) A second nucleotide sequence contained in the GENSET genome sequence and located downstream of the first GENSET nucleotide sequence (a) on the genome. In a preferred embodiment, the DNA construct is further located containing a negative selectable marker located upstream of nucleotide sequence (a) or downstream of nucleotide sequence (c). Preferably, the negative selectable marker is the thymidine kinase (tk) gene
(Thomas et al., 1986), hygromycin beta gene (Te Riele et al., 1990),
the hprt gene (Van der Lugt et al., 1991; Reid et al., 1990) or the A fragment of diphtheria toxin (Dt-A) gene (Nada et al., 1993; Yagi et al., 1990), the disclosures of which are fully incorporated herein. Are incorporated by reference. Preferably, the positive selectable marker is GE so as to interrupt the sequence encoding the GENSET protein.
Located within the NSET exon array. These replacement vectors are described, for example, in Thomas et al. (1986
1987), Mansour et al. (1988) and Koller et al. (1992). The first and second nucleotide sequences (a) and (c) are independent of each other, the GENSET control sequence,
Intron sequences, exon sequences, or regulatory and / or introns and / or
Or it can be located in a sequence containing both exon sequences. Nucleotide sequence
The size of (a) and (c) is in the range of 1-50 kb, preferably 1-10 kb
It is preferably 2 to 6 kb, and most preferably 2 to 4 kb. [DNA construct that enables homologous recombination: Cre-LoxP system] These new DNA constructs utilize the site-specific recombination system of P1 phage. The P1 phage specifically interacts with the 34 base pair loxP site, Cre.
It has a recombinant enzyme called. The loxP site consists of two 13 bp palindromic sequences separated by a 8 bp conserved sequence (Hoess et al., 1986), the disclosure of which is incorporated herein by reference in its entirety. The Cre-loxP system used in conjunction with the homologous recombination technique was described by Gu et al. (1993, 1994), the disclosure of which is incorporated herein by reference in its entirety. That is, the nucleotide sequence of interest, which should be inserted at the target location in the genome, is in a similar orientation and contains at least two loxP sites located at each end of the nucleotide sequence to be excised from the recombinant genome. . The event of ablation is
It requires the presence of the recombinant enzyme (Cre) in the nucleus of the recombinant cell host. The recombinant enzyme can be introduced at a desired time point as follows. (a) by directly injecting the Cre enzyme into the desired cells, as described by Araki et al. (1995), the disclosure of which is incorporated herein by reference, or its publication. By lipofection of the enzyme into cells as described by Baubonis et al. (1993), which is incorporated herein by reference in its entirety, to give recombinant cell hosts in culture medium containing the enzyme. Incubate. (b) Gu et al. (1993) and Sauer et al., whose disclosures are incorporated herein by reference in their entirety.
(1988), a vector containing a Cre coding sequence operably linked to a promoter operable in a recombinant cell host is transfected into the cell host, optionally with the promoter. Inducible and introducing the vector into a recombinant cell host. (c) Introducing a polynucleotide containing a Cre coding sequence operably linked to a promoter operable in a recombinant cell host into the genome of the cell host, as described by Gu et al. (1994). However, the promoter is optionally inducible and the polynucleotide is introduced into the cell host by a random insertion event or homologous recombination event.

In a specific embodiment, the vector containing the sequence to be inserted into the GENSET gene by homologous recombination is constructed so that the selectable marker is flanked by loxP sites in the same orientation, and The treatment allows the selection marker to be deleted while leaving the GENSET sequence of interest introduced by the homologous recombination event.
Again, two selectable markers are needed: a positive selection marker for selecting recombination events and a negative selection marker for selecting homologous recombination events.
Vectors and methods using the Cre-loxP system are described by Zou et al. (1994), the disclosure of which is incorporated herein by reference in its entirety. Thus, a third preferred DNA construct of the invention contains from 5'to 3'end the following: (a) the first nucleotide sequence contained in the GENSET genomic sequence; (b)
A nucleotide sequence containing a polynucleotide encoding a positive selection marker, the nucleotide sequence further contains two sequences that define a site such as a loxP site that can be recognized by a recombinant enzyme, and the two sites are similar. (C) a second nucleotide sequence contained in the GENSET genomic sequence and located downstream of the first GENSET nucleotide sequence (a) in the genome. The site, such as the loxP site, that can be recognized by the recombinant enzyme is preferably located locally in the nucleotide sequence (b) adjacent to the nucleotide sequence sought for conditional excision. In one specific embodiment, the two loxP sites flank the positive selectable marker sequence, allowing its excision at the desired time after the homologous recombination event. In a preferred embodiment of the method using the third DNA construct, excision of a polynucleotide fragment flanked by two sites, preferably two loxP sites, recognized by the recombinant enzyme is carried out at the desired time point. This is done both with a promoter sequence, preferably an inducible promoter, more preferably a tissue-specific promoter sequence, and most preferably both transducible and tissue-specific as described by Gu et al. (1994). Due to the presence in the genome of recombinant host cells of a sequence encoding the Cre enzyme operably linked to a promoter sequence. The presence of the Cre enzyme in the genome of the recombinant host may be due to the breeding of two transgenic animals, where the first transgenic animal is derived from the desired GENSET containing the loxP site as described above. The second transgenic animal has the sequence and the Cre coding sequence operably linked to an appropriate promoter sequence as described by Gu et al. (1994).

In addition, spatial and temporal control of Cre enzyme expression is described by Anton and Graham (1995).
, Kanegae et al. (1995), comprising an adenovirus-based Cre gene for delivery of the Cre enzyme, i.e., achieving by means of a vector capable of infecting cells or organs in vivo. Is possible, the disclosure of which is incorporated by reference in its entirety. The DNA constructs described above should be used to introduce a nucleotide copy of interest at a given position in the target genome, preferably a GENSET genomic sequence or GENSET cDNA sequence, and most preferably an altered copy of the GENSET genomic or cDNA sequence. Can produce an altered copy of the target gene (knockout homologous recombination), or the replacement of the target gene copy with another copy that is sufficiently homologous to allow a homologous recombination event to occur (knock-in homolog Change). Alteration of Expression and / or Biological Activity of GENSET Alteration of endogenous GENSET expression and / or biological activity is specifically contemplated by the present invention. Screening for Compounds Modulating GENSET Expression and / or Biological Activity The present invention also relates to compounds capable of modulating GENSET expression and / or biological activity and methods of using those compounds. Such compounds may interact with the regulatory sequences of the GENSET gene or directly or indirectly with the GENSET polypeptide. <Compounds that interact with GENSET regulatory sequences> The present invention also employs any of the detection methods known to those of skill in the art, including, for example, those disclosed in the section entitled "Identification of promoters for cloned upstream sequences". The present invention relates to a method for screening a substance or molecule capable of interacting with a regulatory sequence of the GENSET gene such as a promoter or enhancer sequence in the untranslated region of genomic DNA determined by the above, or a regulatory sequence located in the untranslated region of GENSET mRNA. The sequence within the non-transcribed or untranslated region of the polynucleotide of the present invention is
, Transcription factor binding site, promoter sequence, enhancer sequence, 5'UTR and 3
'Identified by comparison with databases containing known regulatory sequences such as UTR elements (Pesole et al., 2000; http://igs-server.cnrs-mrs.fr/~gauthere/UTR/index.
html). Alternatively, the control sequence of interest can be identified by conventional mutagenesis or deletion analysis of the reporter plasmid, eg, using the techniques described in the section entitled "Identification of the Promoter of Cloned Upstream Sequences".

After identification of potential GENSET regulatory sequences, proteins that interact with these regulatory sequences can be identified as described below. Fried and Crothers (1981), Garner and Revzin (1981), Dent and L
Gel delay measurements can be performed independently to screen for candidate molecules that interact with the regulatory sequences of the GENSET gene as described by atchman (1993), the teachings of these publications are incorporated herein by reference. Incorporated as a reference. These techniques follow the principle that protein-bound DNA or mRNA fragments migrate slower than the same non-binding DNA and mRNA fragments. That is, the target nucleotide sequence is labeled. The labeled target nucleotide sequence is then contacted with total nuclear extract from cells containing the regulatory factor, or various candidate molecules to be tested. The interaction between the target control sequence of the GENSET gene and the candidate molecule or regulatory factor is detected based on the migration delay after gel or capillary electrophoresis. Nucleic acid encoding a protein capable of interacting with the promoter sequence of the GENSET gene, particularly a nucleotide sequence selected from the group consisting of 5'and 3'regulatory region polynucleotides or fragments or variants thereof, is available from Clontech Ma
It can be identified using a single hybrid system as described in the booklet enclosed in the tchmaker One-Hybrid System kit (catalog reference number n ° K1603-1), the technical teaching of which is incorporated herein by reference. Transferred. That is, the target nucleotide sequence is cloned upstream of the selectable reporter sequence and the resulting polynucleotide construct is integrated into the yeast genome (Saccharomyces cerevisiae). Preferably, multiple copies of the target sequence are inserted in tandem into the reporter plasmid.
Next, a yeast cell containing a reporter sequence in its genome has a fusion molecule consisting of a cDNA encoding a candidate protein that binds to the regulatory sequence of the GENSET gene and a sequence encoding the activation domain of a yeast transcription factor such as GAL4. Transform with the containing library. Recombinant yeast cells are seeded in culture broth to select cells expressing the reporter sequence. The recombinant yeast cells selected in this way are
It contains a fusion protein capable of binding to the target control sequence of the GENSET gene. The cDNAs encoding the fusion proteins can then be sequenced and cloned into expression or transcription vectors in vitro. Binding of the encoded polypeptide to the target control sequence of the GENSET gene can be confirmed using techniques known to those of skill in the art such as gel retardation measurements or DNAse protection assays.

Ligand that Interacts with GENSET Polypeptide For the purposes of the present invention, ligand means binds to a molecule such as a protein, peptide, antibody, or one or a variant of the GENSET protein or fragment thereof. By any synthetic chemical entity capable of or modulating the expression of a polynucleotide encoding GENSET, a fragment or variant thereof. According to the ligand screening method of the present invention, a molecular biological sample to be tested as a putative GENSET protein ligand is produced by a corresponding purified GENSET protein, eg, a recombinant cell host as described herein. The corresponding purified recombinant GENSET protein is contacted and a complex is formed between this protein and the putative ligand molecule to be tested. As a specific example, from GENSET protein or the sequences of SEQ ID NOs: 242-482, the mature polypeptides contained in SEQ ID NOs: 242-272 and 274-384, as well as the full-length or mature polypeptides encoded by the clone inserts in the deposited clone pool. At least 6, preferably at least 8 of the polypeptides selected from the group consisting of
~ 10, more preferably a fragment containing contiguous spans of 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, or 100 amino acids, and produced by the method of drug or combinatorial chemistry. To study interactions with small molecules such as molecules, the microdialysis method combined with the HPLC method described by Wang et al. (1997) or the affinity capillary electrolysis described by Bush et al. (1997). It can be done by electrophoretic methods, the disclosure of which is incorporated by reference. In a further method, the invention provides a GENSET protein or a sequence of SEQ ID NOS: 242-482, a mature polypeptide contained in SEQ ID NOS: 242-272 and 274-384, and a full-length or mature encoded by a clone insert in the deposited clone pool. At least 6, preferably at least 8-10, more preferably 12, 15, 20, 25, 30, 35, 40, 50 of a polypeptide selected from the group consisting of polypeptides,
Alternatively, peptides, drugs, fatty acids, lipoproteins or small molecules that interact with fragments containing contiguous spans of 100 amino acids can be identified by the following assays. The molecule to be tested for binding is labeled with a detectable label such as a fluorescent, radioactive or enzyme tag and contacted with the immobilized GENSET protein or fragment thereof under conditions which allow specific binding. After removing non-specifically bound molecules, bound molecules are detected using suitable means.

A wide variety of candidates or molecules can be assayed for GENSET polypeptide interactions. These substances or molecules include but are not limited to molecules of biological origin, such as natural or synthetic organic compounds or polypeptides. If the candidate substance or molecule comprises a polypeptide, the polypeptide may be the expression product of a phage clone belonging to a phage-based random peptide library. Alternatively, the polypeptide may be the expression product of a cDNA library cloned into a vector suitable for conducting a two-hybrid screening assay. A. Candidate Ligands Obtained from a Random Peptide Library In certain embodiments of the screening method, the putative ligand is the expression product of a DNA insert contained in a phage vector (Parmley and Smith, 1988).
For details, use a random peptide phage library. Random DNA inserts encode peptides of 8-20 amino acids in length (Oldenburg et al., 1992; Valadon et al., 1996; Lucas, 1994; Westerink, 1995; Felici et al., 1991).
, The entire text of which is incorporated herein by reference. In accordance with this particular embodiment, recombinant phage expressing a protein that binds to immobilized GENSET protein are retained, and a complex of GENSET protein and recombinant phage is subsequently cloned or cloned against the GENSET protein. Can be immunoprecipitated with antibody. After making the ligand library in recombinant phage, the phage population is contacted with the immobilized GENSET protein. The complex preparation is then washed to remove non-specifically bound recombinant phage. Phages that specifically bind to the GENSET protein were then eluted with buffer (acidic pH), or immunoprecipitated with a monoclonal antibody produced by anti-GENSET hybridomas, and the phage population was subsequently cloned into bacteria.
Amplify by overinfection with E. coli. The selection step can be repeated several times, preferably 2-4 times, in order to select the most specific recombinant phage clones. The final step is to express the peptide produced by the clone of the selected recombinant phage in an infecting bacterium and then isolate it, or to express the phage insert in another host vector system or to select the recombinant phage. Consisting of characterizing the included insert by sequencing.

[B. Candidate Ligands Obtained by Competitive Experiment] Alternatively, the GENSET protein or the sequences of SEQ ID NOs: 242-482 and SEQ ID NOs: 242-1
72 and 274-384, and at least 6, preferably at least 8-10, more preferably a polypeptide selected from the group consisting of the mature polypeptide and the full-length or mature polypeptide encoded by the clone insert in the deposited clone pool. Can identify peptides, drugs, small molecules that bind to fragments containing contiguous spans of 12, 15, 20, 25, 30, 35, 40, 50 amino acids in competition experiments. In such assays, the GENSET protein, or fragment thereof, is immobilized on a surface such as a plastic plate. Increasing amounts of peptide, drug or small molecule are contacted with the immobilized GENSET protein or fragment thereof in the presence of a detectably labeled known GENSET protein ligand. For example,
GENSET ligands can be detectably labeled with fluorescent, radioactive, or enzyme tags. The ability of a test molecule to bind to the GENSET protein or fragment thereof can be determined by measuring the amount of a detectably labeled known ligand bound in the presence of the test molecule. When a test molecule is present, the decrease in the amount of known ligand bound to the GENSET protein or fragment thereof is due to the test molecule
It shows that it can bind to a protein or a fragment thereof. [C. Candidate Ligands Obtained by Affinity Chromatography] GENSET protein or sequences of SEQ ID NOs: 242-482, SEQ ID NOs: 242-272 and
274-384 mature polypeptide, and at least 6, preferably at least 8-10, more preferably a polypeptide selected from the group consisting of full-length or mature polypeptides encoded by clone inserts in the deposited clone pool. Other molecules that interact with fragments containing contiguous spans of 12, 15, 20, 25, 30, 35, 40, 50, or 100 amino acids can be detected on an affinity column containing the GENSET protein or fragments thereof. The GENSET protein or fragment thereof is immobilized on a column using conventional techniques, including chemical coupling, to a suitable column matrix, such as agarose, Affi Gel (r), or other matrices known to those of skill in the art. it can. In some of the embodiments of this method, the affinity column comprises a chimeric protein in which the GENSET protein or fragment thereof is fused to glutathione S-transferase (GST). The mixture of cellular proteins or pool of expressed proteins is injected onto the affinity column as described above. Proteins or other molecules that interact with GENSET protein or fragments thereof immobilized on a column can then be isolated and analyzed by two-dimensional gel electrophoresis, which is described by Ramunsen et al. (1997), the disclosure of which is Incorporated as a reference. Alternatively, the proteins retained on the affinity column can be purified and sequenced by electrophoresis-based methods. Antibodies can be isolated using the same method to screen phage display products or to screen phage display human antibodies.

[D. Candidate Ligands Obtained by Optical Biosensor Method] GENSET protein or sequences of SEQ ID NOs: 242-482, SEQ ID NOs: 242-272 and
274-384 mature polypeptide, and at least 6, preferably at least 8-10, more preferably a polypeptide selected from the group consisting of full-length or mature polypeptides encoded by clone inserts in the deposited clone pool. Proteins that interact with fragments containing contiguous spans of 12, 15, 20, 25, 30, 35, 40, 50, or 100 amino acids are Edwards and Lea.
therbarrow (1997) and can be screened using optical biosensors as described in Szabo et al. (1995), the disclosure of which is incorporated by reference. This technique does not require labeled molecules and can detect interactions between molecules in real time. This technique is based on the phenomenon of surface plasmon resonance (SPR). That is, the candidate ligand molecule to be tested is immobilized on the surface (such as a carboxymethyldextran matrix). A beam of light is directed toward and reflected by the surface that does not contain the sample to be tested. The intensity of light reflected by the SPR phenomenon decreases with a specific combination of angle and wavelength. Binding of the candidate ligand molecule changes the refractive index of the surface, which is detected as a change in the SPR signal. The GENSET protein or fragment thereof is immobilized on the surface to screen for candidate ligand molecules or substances capable of interacting with the GENSET protein or fragment thereof. This surface comprises one side of the cell through which the candidate molecule to be assayed flows. Binding of the candidate molecule to the GENSET protein or fragment thereof is detected as a variation in the SPR signal. Candidate molecules to be tested may be proteins, peptides, carbohydrates, lipids or small molecules produced by combinatorial chemistry. In addition, this technique may be performed by immobilizing eukaryotic or prokaryotic cells or lipid vesicles, which display on their surface the endogenous or recombinantly expressed GENSET protein. The main advantage of this method is that it allows determination of the association rate between the GENSET protein and the molecules that interact with it. Therefore, it is possible to specifically select a ligand molecule that interacts with the GENSET protein or a fragment thereof via a strong or, conversely, a weak association constant.

E. Candidate Ligands Obtained Through Two-Hybrid Screening Assays The yeast two-hybrid system was designed to study protein-protein interactions in vivo (Fields and Song, 1989), the entire disclosure of which is incorporated herein by reference and is based on the fusion of the bait protein and the DNA binding domain of the yeast Gal4 protein. This technique is further described in US Pat. No. 5,667,973 and US Pat. No. 5,283,173, the technical teachings of both patents are incorporated herein by reference. The general library screening process by the two-hybrid assay is Harp
er et al. (1993) or Cho et al. (1998) or Fromont-Racine et al.
1997), and their disclosures are incorporated herein by reference in their entireties. The bait protein or polypeptide is a GENSET polypeptide or a mature polypeptide contained in the sequences of SEQ ID NOs: 242-482, SEQ ID NOS: 242-272 and 274-384, as well as full-length or mature encoded by a clone insert in the deposited clone pool. At least a polypeptide selected from the group consisting of polypeptides
6, preferably at least 8-10, more preferably 12, 15, 20, 25, 30, 35.
From a fragment containing contiguous spans of 40, 50, or 100 amino acids,
Consist, consist predominantly, or include. More specifically, the nucleotide sequence encoding the GENSET polypeptide or fragment or variant thereof is fused to a polynucleotide encoding the DNA binding region of the GAL4 protein, the fused nucleotide sequence being a suitable expression vector, such as It has been inserted into pAS2 or pM3. Next, a human cDNA library is created in a vector specifically designed to fuse the human cDNA insert with the nucleotide sequence of the vector encoding the transcriptional domain of the GAL4 protein. Preferably the vector used is the pACT vector. The polypeptide encoded by the nucleotide insert of the human cDNA library is referred to as the "prey" polypeptide. The third vector contains a detectable marker gene such as the beta-galactosidase gene or the CAT gene under the control of regulatory sequences that respond to binding of the complete Gal4 protein containing both the transcriptional activation domain and the DNA binding region. To do. For example, the pG5EC vector may be used.

In addition, two different yeast strains are used. As a specific but non-limiting example, the two different yeast strains may be: -Y190, the phenotype is (MATa, Leu2-3, 112 ura3-12, trp1-901, his3-D200, ade2-10
1, gal4Dgal180D URA3 GAL-LacZ, LYS GAL-HIS3, cyhr);-Y187, its phenotype (MATa gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3
, -112 URA3 GAL-lacZmet-), which is a mating type opposite to Y190. That is, 20 μg of pAS2 / GENSET and 20 μg of pACT-cDNA library were added to the yeast strain.
Cotransform to Y190. Transformants are selected for growth on minimal medium lacking histidine, leucine and tryptophan, but containing the histidine synthesis inhibitor 3-AT (50 mM). Positive colonies are screened for beta-galactosidase by filter lift assay. Double-positive colonies (His +, beta-gal +) were then cultured on plates lacking histidine, leucine but containing tryptophan and cycloheximide (10 mg / ml) and pAS2
Selection for deletion of the / GENSET plasmid, but retention of the pACT-cDNA library plasmid. The resulting Y190 strain is GENSET or cyclophilin B,
Y expressing an unrelated control protein such as lamin or SNF1 as a Gal4 fusion
The 187 strain is mated and this is done as described by Harper et al. (1993) and Bram et al. (1993), the publication of which is incorporated by reference herein in its entirety and by filter lift assay. Screen for beta-galactosidase. A yeast clone of beta gal- after mating with a control Gal4 fusion is considered a false positive. In another embodiment of the two-hybrid method of the invention, the interaction of GENSET or a fragment or variant thereof with a cellular protein is the Matchmaker Two Hybrid Sy.
It can be evaluated by using stem 2 (Catalog No. K1604-1, Clontech). As described in the manual attached to the kit, the nucleic acid encoding the GENSET protein or a part thereof is inserted into an expression vector together with the frame of DNA encoding the DNA binding domain of the yeast transcriptional activator GAL4, and the The entire disclosure is incorporated by reference into the present specification. The desired cDNA, preferably human cDNA, is inserted into the second expression vector in frame with DNA encoding the activation region of GAL4. Both expression plasmids are transformed into yeast, which are seeded on a selective medium that is selectable for expression of the selectable marker of each expression vector as well as expression of the GAL4-dependent HIS3 gene. Histidine-deficient media-foldable transformants are screened for GAL4-dependent lacZ expression. Histidine selection and la
Cells positive in both cZ assays are those that contain an interaction between GENSET and the protein or peptide encoded by the initially selected cDNA insert.

Compounds that Modulate GENSET Biological Activity Other methods of screening for compounds that modulate GENSET gene expression and / or biological activity include testing compounds in a host such as apoptosis, proliferation, differentiation, glycosylation of proteins, etc. The effect on the predetermined cellular properties of the cells can be achieved by various techniques known to those skilled in the art, including those disclosed herein, particularly those disclosed in the section entitled "Erreur! Source du renvoi introuvable." It is something to measure. In one embodiment, the present invention relates to a method of identifying a formulation that alters GENSET activity, which comprises introducing into a host cell a nucleic acid construct containing a nucleic acid encoding a mammalian GENSET polypeptide. The resulting host cell is an encoded mammalian GENSET.
The polypeptide is maintained under suitable conditions for expression, which results in expression of the nucleic acid. The host cells are then contacted with the compound (formulation) to be evaluated and the predetermined cellular properties of the cells are detected in the presence of the compound to be evaluated. The detection of a change in certain cellular properties in the presence of the formulation indicates that the formulation alters GENSET activity. In a particular embodiment, the invention is characterized in that the formulation activates GENSET activity by detecting a change in certain cellular properties in the presence of the formulation.
It relates to a method for identifying a preparation which is an activator of GENSET activity. In certain embodiments, the invention provides for the detection of a change in a predetermined cellular property in the presence of a formulation,
It relates to a method for identifying a preparation which is a suppressor of GENSET activity, which is characterized by showing that the preparation suppresses GENSET activity. <Methods for Screening Compounds that Modulate GENSET Expression and / or Biological Activity> The present invention also relates to methods for screening compounds for the ability to modulate (eg, increase or inhibit) activity or expression of the GENSET gene.
More particularly, the invention relates to methods of testing compounds for their ability to either increase or decrease GENSET expression or activity. The assay is performed in vitro or in vivo. [In Vitro Method] GENSET-expressing cells are incubated in vitro in the presence and absence of a test compound. By determining the level of GENSET expression in the presence of test compound or the level of GENSET activity in the presence of test compound, compounds that suppress or amplify GENSET expression or activity can be identified. Instead, reporter genes (eg luciferase, chloramphenicol acetyltransferase, La
Constructs containing a GENSET control sequence operably linked to cZ, green fluorescent protein, etc.) can be introduced into host cells to detect the effect of test compound on reporter gene expression. Suitable cells for use in the above assay are listed in
It includes, but is not limited to, cells of the same origin as the tissue or cell line in which the polypeptide is known to be expressed using the IX data.

The invention thus includes a method of screening for molecules that modulate the expression of the GENSET gene, which method comprises the steps of: a) the GENSET protein or variant or fragment thereof as its own promoter. Culturing a prokaryotic or eukaryotic cell transfected with a nucleotide sequence encoding under the control of b) contacting the molecule to be tested with said cultured cell, c) said GENSET protein or variant or fragment thereof. Expression is quantified in the presence of the molecule. The DNA sequence encoding the GENSET protein is inserted into the expression vector, downstream of its promoter sequence, using DNA recombination techniques known to those of skill in the art.
As a specific example, the promoter sequence of the GENSET gene is 5'of GENSET genomic DNA.
It is included in the non-transcribed region. Quantification of GENSET protein expression can be achieved by measuring mRNA levels (eg GENSET mR of interest).
Northern blot, RT-P with primers and probes specific for NA
CR, preferably using quantitative RT-PCR, or protein levels (using polyclonal or monoclonal antibodies in immunoassays such as ELISA or RIA assays, Western blotting, or immunochemistry) can be performed. The present invention further relates to a method for screening a substance or molecule capable of increasing or conversely decreasing the expression level of the GENSET gene. By this method, GENSET
Those skilled in the art will find substances that exert a regulatory effect on the expression level of genes and may be useful as active ingredients in pharmaceutical compositions for treating patients suffering from disorders associated with abnormal levels of GENSET products. May be able to choose. Thus, part of the invention is a method of screening for candidate molecules that modulate the expression of the GENSET gene, which method comprises the steps of: a) including a nucleic acid, wherein the nucleic acid encodes a detectable protein. Providing a recombinant cell host comprising a GENSET 5'regulatory region operably linked to a polynucleotide or a regulatory active fragment or variant thereof, b) obtaining a candidate molecule, and c) adding the detectable protein. The ability of the candidate molecule to modulate the expression level of the encoding polynucleotide is determined.

In a further embodiment, the nucleic acid comprising the GENSET 5'regulatory region or regulatory active fragment or variant thereof is the sequence of SEQ ID NOs: 1-241, the sequence of the clone insert of the deposited clone pool, the regulatory active fragment thereof. And of the mutant
Contains the 5'UTR region of GENSET cDNA selected from the group containing 5'UTR. In a further preferred embodiment of the above screening method, the nucleic acid contains a promoter sequence endogenous to the GENSET 5'UTR sequence. In yet another preferred embodiment of the above screening method, the nucleic acid contains a promoter sequence exogenous to the GENSET 5'UTR sequence. Preferred polynucleotides encoding detectable proteins are those encoding beta-galactosidase, green fluorescent protein (GFP) and chloramphenicol acetyl transferase (CAT). The invention further comprises a method of screening for candidate molecules that modulate the expression of the GENSET gene, and further mixing the identified molecules with a pharmacologically acceptable carrier.
A method of producing a pharmaceutical composition. The present invention further relates to a kit for screening candidate substances that modulate the expression of the GENSET gene. Preferably, such kits provide for expression of a GENSET 5'regulatory region or regulatory active fragment or variant thereof operably linked to a polynucleotide encoding a detectable protein or GENSET protein or fragment or variant thereof. Includes a recombinant vector that enables it. More preferably, such a kit comprises a sequence of SEQ ID NOs: 1-241 operatively linked to a polynucleotide encoding a detectable protein, the sequence of a clone insert of the deposited clone pool, their regulatory active fragments and It comprises a recombinant vector containing a nucleic acid containing the 5'UTR region of a GENSET cDNA selected from the group containing the mutant 5'UTR. To design suitable recombinant vectors useful in performing the screening methods described above, reference is made to the section describing preferred recombinant vectors of the invention herein. Other objects of the invention include methods and kits for screening candidate agents that interact with the GENSET polypeptide or fragments or variants thereof.
The ability to bind covalently or non-covalently to the GENSET protein or fragments or variants thereof allows these agents or molecules to be conveniently used in vitro and in vivo.

In vitro, the interacting molecule is present in a sample, preferably a biological sample.
It can be used as a detection tool to identify the presence of the GENSET protein. A method of screening a candidate substance that interacts with a GENSET polypeptide or fragment or variant thereof, which method comprises the steps of: a) a GENSET protein or a sequence of SEQ ID NOs: 242-482, SEQ ID NOs: 242-272 and 274 ~ 384 at least 6, preferably at least 8-10, more preferably at least 6 of the polypeptides selected from the group consisting of the full-length or mature polypeptides encoded by the clone inserts of the deposited clone pool. Providing a polypeptide consisting of, consisting predominantly of, or comprising, a contiguous span of 15, 20, 25, 30, 35, 40, 50, or 100 amino acids, b) obtaining a candidate molecule, c) said Contacting the polypeptide with the candidate substance, and d) detecting a complex formed between the polypeptide and the candidate substance. The present invention further comprises a method of screening a candidate substance that interacts with a GENSET polypeptide or fragment or variant thereof, and further mixing the identified molecule with a pharmacologically acceptable carrier. It relates to a method of producing a thing. The invention further relates to a kit for screening candidate substances that interact with the GENSET polypeptide, which kit comprises: a) GENSET protein or the sequence of SEQ ID NOs: 242-482, SEQ ID NOS: 242-272 and 274. ~ 384 at least 6, preferably at least 8-10, more preferably at least 6 of the polypeptides selected from the group consisting of full-length or mature polypeptides encoded by clone inserts in the deposited clone pool. , 15, 20, 25, 30, 35, 40, 50, or 100 contiguous spans of amino acids, and b) optionally useful said polypeptide or a polypeptide thereof. Means for detecting the complex formed by the mutant and the candidate substance. In a preferred embodiment of the above kit, Detecting means comprises a monoclonal or polyclonal antibody that binds to said GENSET protein or fragment or variant thereof.

In Vivo Method Compounds that suppress or amplify GENSET expression can also be identified using in vivo screening. In these assays, the test compound is administered to the animal, eg, at various dose levels (eg, IV, IP, IM, orally, or otherwise). The effect of a compound on GENSET expression can be determined, for example, by using the data obtained in Table IX for GENSET levels in tissues known to express the gene of interest, and Northern blotting, immunoassays, It is measured by comparison using the PCR method as described above. Suitable test animals include rodents (eg mice and rats), primates, mammals. Humanized mice can also be used as test animals. This is because the endogenous mouse proteins have been removed (knocked out)
And the homologous human protein has been re-added by conventional transgenic methods. Such mice express only human-shaped proteins. Humanized mice expressing only human GENSET have GENSET protein or m
It can be used to study the response to potential formulations that regulate RNA levels in vivo. As an example, a transgenic mouse carrying the human apoE4 gene was created. We then breed with mouse strains lacking endogenous apoE to create animal models that carry human proteins that appear to play a role in the development of Alzheimer's disease states. Such transgenic animals are useful for analyzing the biochemical and physiological steps of the disease and for the development of treatments for disease intervention (Loring, et al., 1996) (referenced herein in its entirety). To be incorporated as). Uses of compounds that modulate GENSET expression and / or biological activity Use in vivo (or in vitro) systems, eg GEN only in tissues of interest
It is possible to identify compounds that exert a tissue-specific effect that increases SET expression or activity. Screening procedures such as those described above are further useful for identifying formulations and their potential use in pharmacological intervention strategies. Agents that amplify GENSET expression or stimulate its activity can be used to treat disorders that require GENSET gene expression and / or activity at upregulated levels. Compounds that suppress GENSET expression or inhibit its activity can be used to treat disorders that require down-regulated levels of GENSET gene expression and / or activity.

The present invention also includes formulations that interact with GENSET directly or indirectly to suppress or increase GENSET expression and / or function. In one embodiment, the agent is an inhibitor that interferes with GENSET directly (eg, by binding to GENSET) or indirectly (eg, by blocking GENSET's ability to have GENSET biological activity). Is. In certain embodiments, an inhibitor of a GENSET protein is an antibody specifically directed against the GENSET protein or a functional portion of GENSET, ie, an antibody that binds GENSET polypeptide binding. For example, the antibody may be a human GENSET gene (a mature polypeptide contained in SEQ ID NOS: 242-482, SEQ ID NOS: 242-272 and 274-384, a full-length or mature polypeptide encoded by a clone insert of the deposited clone pool), a mammalian GENSET or It may be specific for a polypeptide encoded by one of its amino acid sequences. Alternatively, the inhibitor may be a formulation other than an antibody that binds to GENSET and blocks its activity (eg, a small organic molecule, protein or peptide). For example, the inhibitor may be a formulation that structurally mimics GENSET but lacks its function. Alternatively, even a formulation that binds or interacts with a molecule to which GENSET normally binds or interacts, thereby blocking GENSET from doing so and preventing GENSET from exerting the effects that GENSET should normally have. Good. In other embodiments, the formulation increases, either directly or indirectly, the activity of GENSET (increasing the effect of a given amount or level of GENSET), increasing the length of time that GENSET is effective. To prevent its degradation, or by other means
ET is an enhancer (activator) of GENSET, or by prolonging the time that ET is activated. The GENSET sequences of the present invention can also be used to generate non-human gene knockout animals, such as mice lacking the GENSET gene or transgenically overexpressing GENSET. For example, such GENSET gene knockout mice can be generated to further understand the function of GENSET and to assess the specificity of GENSET activators and inhibitors. Furthermore, overexpression of GENSET (eg human GENSET) in transgenic mice can be used as a means of creating a test system for GENSET activators and inhibitors (eg against human GENSET). In addition, the GENSET gene can be used for GENSET promoter / enhancer cloning to identify regulators of GENSET transcription. GENSET gene knockout animals include animals that completely or partially lack the GENSET gene and / or GENSET activity or function. Accordingly, the invention includes administering to a mammal an effective amount of an inhibitor of GENSET,
It relates to methods of (generally or partially) inhibiting GENSET biological activity in mammals (eg humans). The invention further relates to methods of increasing GENSET biological activity in a mammal, including administering to the mammal an effective amount of the enhancer GENSET.

Suppression of GENSET Expression Therapeutic compositions of the present invention conveniently employ one or more GENSET oligonucleotide fragments as antisense or triple helix tool tools to suppress expression of the corresponding GENSET gene. May be included. <Antisense Method> In the antisense method, a nucleic acid sequence complementary to the mRNA is hybridized with the mRNA in the cell, thereby blocking the expression of the protein encoded by the mRNA.
Antisense nucleic acid molecules to be used in gene therapy may be DNA or RNA sequences. A preferred method using an antisense polynucleotide according to the invention is Sc
zakiel et al. (1995), the disclosure of which is incorporated herein by reference in its entirety. Preferably, the antisense tool is GENSET mRNA, more preferably GENSET
It is selected from the polynucleotides (15-200 bp in length) complementary to the 5'end of mRNA. In other embodiments, a combination of different antisense polynucleotides complementary to different desired targeting genes is used. Another preferred antisense polynucleotide of the invention is the translation initiation codon ATG.
Sequence of GENSET mRNA containing, or G containing splicing donor or acceptor site
It is a sequence complementary to one of the sequences of ENSET genomic DNA. Preferably, the antisense polynucleotide of the invention is RNA polymerase II.
The 3'polyadenylation signals were replaced by self-cleaving ribozyme sequences so that transcripts were generated from their 3'end in the absence of poly (A), and these antisense polynucleotides were prepared by Liu et al. 1994) and could not be exported from the nucleus, the disclosure of which is incorporated herein by reference in its entirety. In a preferred embodiment, these GENSET antisense polynucleotides further comprise a histone stem-loop structure within the ribozyme cassette that stabilizes the cleaved transcript against 3'-5 'exonuclease degradation, which structure is described by Eckner et al.
(1991), the disclosure of which is incorporated herein by reference in its entirety. The length and melting temperature of the antisense nucleic acid are sufficient to allow the formation of an intracellular duplex, and the stability of the duplex inhibits the expression of GENSET mRNA in that duplex. Is enough to be done. Strategies for designing suitable antisense nucleic acids for use in gene therapy are described by Green et al. (1986) and Izant and
Weintraub, (1984), the entire disclosure of which is incorporated herein by reference.

In some strategies, antisense molecules are obtained by reversing the orientation of the GENSET translation region with respect to the promoter, which causes it to transcribe the opposite strand of that normally transcribed in cells. Antisense molecules may be transcribed using an in vitro transcription system such as those that use T7 or SP6 polymerase to generate transcripts. Another method involves transcribing the GENSET antisense nucleic acid in vivo by operably linking DNA containing the antisense sequence to a promoter in a suitable expression vector. Alternatively, the oligonucleotide complementary to the strand normally transcribed in the cell may be synthesized in vitro. Thus, antisense nucleic acids are complementary to the corresponding mRNA and are capable of hybridizing with the mRNA and forming duplexes. In some embodiments, the antisense sequences include modified sugar phosphate backbones to increase stability and reduce sensitivity to RNase activity. Examples of modifications suitable for use in antisense strategies include 2'O-methyl RNA oligonucleotides and protein-nucleic acid (PNA) oligonucleotides. Further examples can be found in Rossi et al. (19
91), the entire disclosure of which is incorporated herein by reference. Multiple types of antisense complementary to GENSET cDNA or genomic DNA sequences
Oligonucleotides may be used. In one preferred embodiment, the stable and semi-stable antisense oligonucleotides described in International Application PCT WO94 / 23026, incorporated herein by reference, are used. In these molecules, the 3'end or both the 3'and 5'ends are involved in intramolecular hydrogen bonding between complementary base pairs. These molecules are well tolerated by exonuclease attack and exhibit increased stability as compared to conventional antisense oligonucleotides. In another preferred embodiment, antisense oligodeoxynucleotides against herpes simplex virus types 1 and 2 described in International Application WO 95/04141, which is incorporated herein by reference, are used. In yet another preferred embodiment, the covalently cross-linked antisense oligonucleotides described in International Application WO 96/31523, which is incorporated herein by reference, are used. These double-stranded or single-stranded oligonucleotides each contain a covalent cross-link between or within the oligonucleotides, and the bond is a primary amine group on one strand and a carboxyl group on the other strand or the same strand, respectively. Consisting of an amide bond between and, the primary amine group is directly substituted at the 2'position of the monosaccharide ring of the chain nucleotide, and the carboxyl group is substituted on the other chain or the nucleotide or nucleotide analogue of the same chain, respectively. Carried by the aliphatic spacer group.

The antisense oligodeoxynucleotides and oligonucleotides disclosed in International Application WO 92/18522, incorporated by reference, may also be used. These molecules are stable to degradation and contain at least one transcriptional regulatory recognition sequence that binds regulatory proteins and is effective as a bait. These molecules may include “hairpin” structures, “dumbbell” structures, “modified dumbbell” structures, “crosslink” bait structures and “loop” structures. In another preferred embodiment, the cyclic double-stranded oligonucleotides described in European Patent Application 0 572 287 A2, which is incorporated herein by reference, are used.
These linking oligonucleotides "dumbbells" contain transcription factor binding sites and sequester the factors, thereby inhibiting the expression of genes under the control of the transcription factors. Uses of the closed antisense oligonucleotides disclosed in International Application WO 92/1973, incorporated herein by reference, are also planned. Due to the absence of free ends in these molecules, they are more resistant to degradation by exonucleases than conventional oligonucleotides. These oligonucleotides are multifunctional and may interact with multiple regions that are not adjacent to the target mRNA. Appropriate levels of antisense nucleic acid required to inhibit gene expression may be determined using in vitro expression analysis methods. Antisense molecules may be introduced intracellularly by diffusion, injection, infection or transfection using procedures known in the art. For example, antisense nucleic acids can be neat or naked oligonucleotides,
It can be introduced into the body as an oligonucleotide encapsulated in a lipid, an oligonucleotide sequence in a capsid by a viral protein, or an oligonucleotide operably linked to a promoter contained in an expression vector. The expression vector may be any of various expression vectors known in the art,
Includes retroviral or viral vectors, extrachromosomally replicable vectors, or integrative vectors. The vector may be DNA or RNA. Antisense molecules are present in several different concentrations, preferably 1x10-10M to 1x10-4M.
Is introduced on the cell sample. Once the minimum concentration at which gene expression can be adequately controlled has been identified, the optimized dose is converted to a dose suitable for in vivo use. For example, an inhibitory concentration of 1x10-7 in culture translates to a dose of about 0.6 mg / kg body weight. 100 mg /
Levels of oligonucleotide near or above kg body weight are possible after testing the toxicity of the oligonucleotide in laboratory animals. Furthermore, it is also planned that vertebrate cells be removed, treated with antisense oligonucleotides and reintroduced into vertebrates.

In a preferred application of the invention, the effectiveness of antisense suppression on translation is measured by RIA.
And the polypeptide encoding the gene is first identified so that it can be monitored using techniques including, but not limited to, antibody-mediated tests such as ELISA, functional assays or radiolabeling. An alternative to antisense technology used in accordance with the present invention is a ribozyme that binds to a target sequence via a complementary polynucleotide tail and cleaves the corresponding RNA by hydrolyzing its target site (ie, a "hammerhead ribozyme"). ) Is used. So the simplified cycle of the hammerhead ribozyme is (
1) Sequence-specific binding to target RNA via complementary antisense sequences, (2) Site-specific hydrolysis of cleavable motifs on the target strand, and (3) Release of cleavage products that result in additional catalytic cycles. ,including. Indeed, the use of long antisense polynucleotides (at least 30 bases in length) or ribozymes with long antisense arms would be advantageous. A preferred delivery system for antisense ribozymes is achieved by covalently linking these antisense ribozymes with lipophilic groups or by using liposomes as a convenient vector. Preferred antisense ribozymes of the present invention have been prepared as described by Rossi et al. (1991) and Sczakiel et al. (1995), the specific preparative procedure being incorporated herein by reference. Is referred to in. <Triple helix method> GENSET genomic DNA can also be used to inhibit the expression of the GENSET gene based on intracellular triple helix formation. Triple helix oligonucleotides are used to block transcription from the genome. These triple helix oligonucleotides are particularly useful for studying changes in cellular activity associated with particular genes. GENSET cDNAs of the present invention
Alternatively, genomic DNAs, or more preferably these fragment sequences, can be used to inhibit gene expression in an individual having a disease associated with expression of a particular gene. Similarly, a portion of GENSET genomic DNA is
It can be used to study the effect of suppressing SET transcription. In the past, homopurine sequences appeared to be the most useful for triple helix strategy. However, homopyrimidine sequences can also inhibit gene expression. Such homopyrimidin oligonucleotides bind in the major groove of the homopurine: homopyrimidin sequence. Therefore, both types of sequences of GENSET genomic DNA are contemplated within the scope of the present invention.

To perform gene therapy strategies using the triple helix method, use GENSET
Genomic DNA sequences can be first scanned to identify 10-mer to 20-mer homopyrimidine or homopurine regions and used in triple helix-based strategies to suppress GENSET expression. After identifying a candidate homopyrimidin or homopurine region, the effect of suppressing GENSET expression of the candidate homopyrimidin or homopurine region is evaluated by inserting oligonucleotides containing different amounts of the candidate sequence into tissue culture cells expressing the GENSET gene. . Oligonucleotides can be introduced into cells using a variety of methods known to those of skill in the art including, but not limited to, calcium phosphate precipitation, DEAE-dextran, electroporation, liposome-mediated transfection or natural uptake. Treated cells with Northern blot, RNAse protection assay, or
Techniques that monitor transcription levels of the GENSET gene in oligonucleotide-treated cells, such as PCR-based strategies, are monitored for altered cell function or reduced GENSET expression. The cellular function to be monitored is predicted based on the homology between the target gene corresponding to the cDNA from which the oligonucleotide was derived and the sequence of genes known to be associated with the particular function. “Identification of a gene associated with a genetic disease or drug response” based on the presence of abnormal physiology in cells derived from an individual with a specific genetic disease, especially if the cDNA is associated with the disease. It can also be predicted using the techniques described in the section entitled. Oligonucleotides that are effective in suppressing gene expression in tissue culture cells are:
These techniques can then be used to introduce in vivo at a dose calculated based on the in vitro results, as described in the section entitled "Antisense Methods." In some embodiments, the natural (beta) anomer of the oligonucleotide unit can be replaced with an alpha anomer, rendering the oligonucleotide more resistant to nucleases. In addition, an intercalating agent such as ethidium bromide, or the like, can be attached to the 3'fragment of the alpha oligonucleotide to stabilize the triple helix. For information on the generation of oligonucleotides suitable for triple helix formation, see Griffin et al. (1989), which is incorporated herein by this reference.

Treatment of GENSET-Related Disorders The present invention further provides for GENSET by increasing GENSET activity and / or expression.
A method of treating a disease / disorder. The invention further relates to methods of treating GENSET diseases / disorders by reducing GENSET activity and / or expression. These methodologies can be performed with compounds selected using the screening procedures described in this application, and / or with gene therapy and antisense methods conventional and described in this application. Gene therapy can be used to carry out targeted expression of GENSET. The GENSET coding sequence can be cloned into a suitable expression vector and targeted to specific cell types to achieve efficient high level expression. Introduction of the GENSET coding sequence into target cells has been described, for example, by particle-mediated DNA delivery, (Haynes, 1996 and Maurer, 1999), direct injection of naked DNA, (L
evy et al., 1996; and Felgner, 1996), or viral vector doubling transport (Sm
ith et al., 1996, Stone et al., 2000; Wu and Atai, 2000), each of which is incorporated by reference herein in its entirety. Tissue-specific effects can be achieved, for example, by using a viral vector that is tissue-specific for virus-mediated transport, or by using a tissue-specific promoter. Combinatorial methods can also be used to ensure activation of the GENSET coding sequence in the target tissue (Butt and Karathanasis, 1995; Miller and Whe.
lan, 1997), the entire disclosure of which is incorporated herein by reference. GEN
Antisense oligonucleotides complementary to SET mRNA can be used, for example, to selectively reduce or eliminate protein expression at sites of inflammation. More specifically, antisense constructs or antisense oligonucleotides are listed in Table X.
It is possible to inhibit GENSET production in high expressing cells such as those cited in the third column of. Antisense mRNA can be produced by transfecting an expression vector having the GENSET gene sequence, or a part thereof, into a target cell in the antisense direction with respect to the transcription direction. Suitable vectors include viral vectors, including retroviral, adenoviral, and adeno-associated viral vectors, as well as non-viral vectors. Tissue-specific promoters can be used. Alternatively, antisense oligonucleotides can be introduced directly into target cells to achieve similar goals. (Also refer to other delivery methodologies described in this application related to gene therapy.) Oligonucleotides can be selected / designed to achieve high levels of specificity (Wagner et al., 1996. ),
The entire disclosure of which is incorporated herein by reference. The therapeutic methodologies described in this application are applicable to both human and non-human (including cat and dog) mammals.

(Pharmacologically and Physiologically Acceptable Composition) The present invention also provides a pharmacologically or physiologically acceptable composition containing the polypeptide, nucleic acid or antibody of the present invention as an active preparation. Regarding The invention also relates to compositions comprising as active preparation the compounds selected by the screening procedure described above. Such compositions comprise the active preparation in combination with a pharmacologically or physiologically tolerable carrier. In the case of naked DNA, the "carrier" may be a gold particle. The amount of active preparation in the composition may vary depending on the preparation, the patient and the effect sought. Similarly, the dosage regimen may vary depending on the composition and the disease / disorder being treated. Accordingly, the present invention employs any of the screening methods described herein to select an active formulation, compound, substance or molecule and to further identify the identified active formulation, compound, substance or molecule pharmacologically. To a method of producing a pharmaceutical composition, including mixing with a carrier that is tolerable to PEG. The pharmaceutical composition used in the present invention is oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, tongue. It may be administered by any number of routes including, but not limited to, sub-, or rectal means. In addition to the active ingredient, these pharmaceutical compositions are suitable pharmacologically tolerable carriers, and excipients and auxiliaries which facilitate the process of bringing the active compounds into pharmaceutically acceptable preparations. You may contain the carrier containing. For more detailed information on dosage forms or administration techniques, see Remington's Pharmaceutical Sciences (Maac
Available in the latest edition of k Publishing Co. Easton, Pennsylvania). The pharmaceutical composition for oral administration may be in a dosage form using a pharmacologically acceptable carrier known in the art in a dose suitable for oral administration. Carriers such as these are
Allows pharmaceutical compositions in dosage forms such as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for ingestion by a patient. By combining the active compounds with solid excipients, the resulting mixture is ground, if desired, and the mixture of granules is treated, optionally after adding suitable auxiliaries, to give tablets or dragee cores. An oral pharmaceutical preparation can be obtained. Suitable excipients are carbohydrate or protein fillers such as sugars including lactose, sucrose, mannitol, or sorbitol; starches of corn, wheat, rice, potato, or other plants; methylcellulose, hydroxypropylmethylcellulose, or sodium. Cellulose such as carboxymethyl cellulose; gums including Arabic and tragacanth; and proteins such as gelatin and collagen. If desired, degradants or solubilizers such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or salts thereof, such as sodium alginate, can be added.

Dragee cores are used in conjunction with suitable coating agents such as concentrated sugar solutions,
The coating agent may contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, a lacquer solution, and a suitable solvent or mixture of solvents. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to distinguish different quantities of active compound, ie dose. Oral pharmaceutical preparations include push-fit capsules made of gelatin, as well as capsules consisting of soft, sealed gelatin and a coating such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler or binders such as lactose or starch, lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils with or without stabilizers, liquid, or liquid polyethylene glycol. Preparation of medicaments suitable for parenteral administration is carried out in an aqueous solution, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Additionally, suspensions of the active compounds may be prepared as appropriate fatty injection suspensions. Suitable lipophilic solutions or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or formulations which increase the solubility of the compounds and allow for highly concentrated preparations. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. The pharmaceutical compositions of the present invention may be manufactured in a manner known in the art, for example by means of conventional mixing, dissolving, granulating, dragee-making, grinding, emulsifying, encapsulating, entrapping or lyophilizing processes. . The pharmaceutical composition can be provided as salts and can be formed with many acids including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts are more soluble in aqueous or other protic solutions than are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder, containing any or all of the following in the pH range 4.5-5.5 combined with buffer before use: histidine 1-50 mM. , Sucrose 0.1
% -2%, and mannitol 2-7%.

After the pharmaceutical composition is prepared, it can be placed in a suitable container and labeled for treatment of the indicated medical condition. To administer GENSET, such label should state the amount, frequency and mode of administration. Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. Determination of the effective dose is well within the capabilities of those skilled in the art. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays of neoplastic cells or in animal models, usually mice, rabbits, dogs or pigs. The animal model can also be used to determine the appropriate concentration range and route of administration. Such information can be used to determine useful doses and routes for administration in humans. A therapeutically effective dose refers to an active ingredient, such as GE, that ameliorates the symptoms or condition.
The amount of NSET or a fragment thereof, GENSET antibody, GENSET agonist, antagonist or inhibitor is indicated. Therapeutic efficacy and toxicity can be measured in cell cultures or experimental animals by eg ED50 (therapeutically effective dose for 50% of the population).
And LD50 (a dose lethal to 50% of the population) and can be determined by standard pharmacological procedures. The dose ratio between therapeutic and toxic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Pharmaceutical compositions that exhibit high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for humans. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED50 with little or no toxicity. The dosage will vary within this range depending upon the dosage form used, the sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner based on factors related to the patient in need of treatment. Dosage and administration are modulated to provide a sufficient level of active moiety to maintain the desired effect. Factors that can be considered include the severity of the condition, the patient's overall health, age, weight and sex of the patient, dietary habits, time and frequency of administration, drug combination, response sensitivity, and resistance / response to treatment. Long-term effective pharmaceutical compositions may be administered every 3 to 4 days, weekly or biweekly, depending on the half-life and clearance rate of the particular dosage form.

A normal dose varies from 0.1 to 100,000 micrograms, a total dose of about 1 g, depending on the route of administration. Guidance on specific dosages and delivery methods is provided in the literature and generally available to physicians in the art. These skilled artisans use different dosage forms for the nucleotides than the proteins or their inhibitor y. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. (Use of GENSET sequence: computer-related embodiment) As used in the present application, expressions such as "cDNA code of SEQ ID NOS: 1 to 241" refer to sequences of SEQ ID NOS: 1 to 241 and sequences of clone inserts of deposited clone pools,
And fragments thereof, nucleotide sequences homologous to them, and nucleotide sequences of sequences complementary to all the above sequences. Fragments are at least 8, 10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50 of SEQ ID NOs: 1-241,
Includes fragments of SEQ ID NOs: 1-241 containing 75, 100, 150, 200, 300, 400, 500, 1000 or 2000 contiguous nucleotides. Preferably the fragment is SEQ ID NO: 1-
The signal sequences and coding sequences of the mature polypeptides of 31 and 33-143, the polynucleotides described in Tables Va and Vb, the polynucleotides encoding the polynucleotides described in Table VI, the present application as a polypeptide having biological activity. Or a signal sequence or coding sequence for the mature polypeptides of SEQ ID NOs: 1-31 and 33-143, the polynucleotides set forth in Table Va and Table Vb, the table
A polynucleotide encoding the polynucleotide described in VI, at least 8, 10 of the polynucleotides described herein as a polypeptide having biological activity.
, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 5
It includes fragments containing 00, 1000 or 2000 consecutive nucleotides. Sequence number 1-
241 homologous sequences and fragments are at least 99%, 98%, 97% of these sequences.
, Sequences having 96%, 95%, 90%, 85%, 80%, or 75% identity. Identity may be determined using any computer program and parameters described in this application, including BLAST2N with default parameters or any modified parameters. Homologous sequences further include RNA sequences in which uridine replaces thymine in the cDNA code of SEQ ID NOs: 1-241. Homologous sequences may be obtained using any of the procedures described in this application, or may be the result of correcting sequencing errors as described above. Preferably the homologous sequences and fragments of SEQ ID NOs: 1-241 are the signal sequences and coding sequences of the mature polypeptides of SEQ ID NOs: 1-31 and 33-143, the polynucleotides described in Tables Va and Vb, and as regions in Table VI. A polynucleotide encoding the polypeptide fragment described, a polynucleotide described herein as a polynucleotide encoding a polypeptide having biological activity, or the signal sequence of the mature polypeptide of SEQ ID NOs: 1-31 and 33-143, or A coding sequence, a polynucleotide described in Table Va and Table Vb, a polynucleotide encoding a polypeptide fragment described as a region in Table VI, at least 8, 10 of the polynucleotides described herein as biologically active polypeptides. , 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200, 300
, 400, 500, 1000 or 2000 consecutive nucleotides with homology to a polynucleotide. It is understood that the cDNA codes of SEQ ID NOs: 1-241 can be represented in the conventional one-letter format (see Styer, 1995, inside back cover) or in any other format that records nucleotide identity in a sequence. Let's do it.

As used herein, the phrase “polypeptide code of SEQ ID NOS: 242-482” refers to the signal peptides included in SEQ ID NOS: 242-482, SEQ ID NOS: 242-272 and 274-384, SEQ ID NOS242-242. 272 and 274-384, including the mature polypeptides, the full length signal peptide and mature polypeptide sequences encoded by the clone inserts of the deposited clone pool, the homologous polypeptide sequences, or fragments of any of the above sequences. To do. A homologous polypeptide sequence means a signal peptide contained in SEQ ID NOS: 242 to 482, SEQ ID NOS: 242 to 272 and 274 to 384, a mature polypeptide contained in SEQ ID NOS: 242 to 272 and 274 to 384, a deposited clone pool. At least 99%, 98%, 97%, 96%, 95%, 90% with the full length signal peptide and mature polypeptide sequence encoded by the clone insert of
, Polypeptide sequences having 85%, 80%, 75% identity are shown. Identity may be determined using any computer program and parameters described in this application, including FASTA with default parameters or any modified parameters. Homologous sequences may be obtained using any of the procedures described in this application,
Alternatively, it may be the result of correcting the sequencing error as described above. Polypeptide fragments include at least 5, 6, 8 of the polypeptides of SEQ ID NOS: 242-482.
, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100, 150, 200, 250, 300, 3
Contains 50, 400, 450 or 500 consecutive amino acids. Preferably the fragment is a signal peptide comprised in SEQ ID NOs: 242-272 and 274-1384, SEQ ID NOs: 242-272.
And the mature polypeptides included in 274-1384, the polynucleotides described in Tables Va and Vb, the domains described in Table VI, the epitopes described in Table VII, the polypeptides described herein as having biological activity, Or a signal peptide contained in SEQ ID NOs: 242-272 and 274-1384, a mature polypeptide contained in SEQ ID NOs: 242-272 and 274-1384, a polynucleotide described in Table Va and Table Vb, Table VI
Domains described in Table VII, epitopes described in Table VII, at least 5, 10, 15, 20, 25, 30, 35, 40 of the polypeptides described herein as having biological activity.
It includes polypeptides encoded by fragments containing 50, 75, 100, 150, 200, 300 or 400 contiguous amino acids. Represent the polypeptide code of SEQ ID NOs: 242-482 in the conventional one-letter or three-letter form (see inside cover of Styer, 1995) or in any other form that correlates the identity of the polypeptides in the sequence You can understand that you can do it.

Those skilled in the art will appreciate that the nucleic acid code of the invention and the polypeptide code of the invention can be stored, recorded and manipulated on a computer readable and accessible medium. In this application, the words "record" and "remember" refer to the process of storing information on a computer medium. Those of ordinary skill in the art can readily employ any known method of recording information on a computer-readable medium to produce one or more nucleic acid codes of the invention or one or more polypeptide code products of the invention. Can be generated. Another aspect of the invention is a computer readable medium having at least 2, 5, 10, 15, 20, 25, 30, 50, 75, 100, 150 or 200 of the nucleic acid code of the invention recorded thereon. . Another aspect of the invention is a computer readable medium having at least 2, 5, 10, 15, 20, 25, 30, 50, 75, 100, 150 or 200 polypeptide codes of the invention recorded thereon. is there. Computer readable media include magnetically readable media, optically readable media,
Includes electronically readable media and magnetic / optical media. For example, a computer-readable medium is a hard disk, a floppy disk, magnetic tape, CD-
ROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Rea
It may be another type of d Only Memory (ROM) as well as other media known to those skilled in the art. The embodiment of the present invention is a system for operating and processing the sequence information described in the present application,
In particular it includes computer systems. An example of computer system 100 is shown in the block diagram of FIG. As used herein, "computer system" refers to the hardware, software, and data storage components used to analyze the nucleotide sequence of the nucleic acid code of the invention or the amino acid sequence of the polypeptide code of the invention. In one embodiment, computer system 100 is a Sun Enterprise 1000 server (Sun Microsystems, Paolo Alto, CA). Computer system 100 preferably includes a processor for processing, accessing and manipulating sequence data. Processor
105 may be any known type of central processing unit, such as Pentium III from Intel Corporation, or similar processor from Sun, Motorola, Compaq or International Business Machines. Computer system 100 preferably includes processor 10 for data storage.
A multi-purpose system including five and one or more internal data storage components 110 and one or more data retrieval devices for retrieving data stored on the data storage components. Those of ordinary skill in the art will readily appreciate that any of the currently available computer systems are suitable.

In an example of a particular embodiment, computer system 100 includes main memory 115.
A processor connected to the bus that connects (preferably implemented as RAM)
105, and one or more internal data storage devices 110, such as a hard drive and / or other computer-readable medium having data stored thereon. In some embodiments, computer system 100 further includes one or more data retrieval devices 118 for reading the data stored on internal data storage device 110. The data retrieval device 118 may represent, for example, a floppy disk drive, a compact disk drive, an electromagnetic drive, or the like. In some embodiments, internal data storage device 110 is a removable computer-readable medium, such as a floppy disk drive, compact disk drive, electromagnetic drive, containing control logic and / or data stored thereon. .. Computer system 100 may conveniently include or be programmed with suitable software to read control logic and / or data from the data storage component once inserted into the data retrieval device. Computer system 100 includes a display 120 for displaying output to a computer user. It should also be noted that computer system 100 can be connected to computer systems 125a-c on other networks or wide area networks and provide centralized access to computer system 100. Software for accessing and processing the nucleotide sequence of the nucleic acid code or the amino acid sequence of the polypeptide code of the present invention (search tool, comparison tool,
And modeling tools, etc.) may reside in main memory during execution. In some embodiments, computer system 100 further comprises a nucleic acid code or polypeptide of the invention described above stored on a computer readable medium, a reference nucleotide or polypeptide stored on the computer readable medium. A sequence comparer for comparison with the sequence may be included. A "sequence comparer" is a compound that includes, but is not limited to, a nucleotide or polypeptide sequence, other nucleotide or polypeptide sequences stored in data storage means, and / or peptides, peptide analogs, chemicals. For comparison, one or more programs installed in computer system 100 are shown. For example, a sequence comparer compares a nucleotide sequence of a nucleic acid code of the invention or an amino acid sequence of a polypeptide code of the invention stored on a computer readable medium to a reference sequence stored on a computer readable medium. Thus, a homology, a motif involved in biological function, or a structural motif can be identified. The various sequence comparison programs identified elsewhere in this patent specification are specifically designed for use in such aspects of the invention. FIG. 3 is a flow chart showing one embodiment of a process 200 for comparing a novel nucleotide or protein sequence to a sequence database to determine the level of homology between the new sequence and the sequence in the database. The sequence database may be a proprietary database stored in computer system 100 or a public database available via the Internet such as GENBANK, PIR or SWISSPROT.

The process 200 starts at the start state 201 and moves to the state 202 where the new array to be compared is stored in the memory of the computer system 100. As explained above, the memory may be any type of memory including RAM or internal storage devices. Process 200 then moves to state 204 with the sequence database open for analysis and comparison. Process 200 then moves to state 206 where the first array stored in the database is read into computer memory. Next, a comparison is made at state 210 to determine if the first sequence is equivalent to the second sequence. This step
It is important not to be limited to making an exact comparison between the new sequence and the first sequence in the database. Known methods for comparing two nucleotide or protein sequences, even if they are not identical, are known to those skilled in the art. For example, a gap can be introduced in one of the sequences to improve the level of homology between the two sequences tested. The parameters controlling whether gaps or other features are introduced into the sequence during comparison are usually entered by the user of the computer system. Once state 210 is compared for both sequences, decision state 210 determines if both sequences are equivalent. Of course, the term "equivalent" is not limited to exact matching sequences. The sequences within the homology parameters entered by the user are marked as “equivalent” in process 200. If both sequences are determined to be equivalent, the process 200 moves to state 214 where the sequence names in the database are displayed to the user. This state notifies the user that the sequence whose sequence name is displayed satisfies the entered homology constraint. Once the stored sequence name is displayed to the user, process 200 moves to state 218, which determines if there are more sequences in the database. If no more arrays are present in the database, process 200 ends in end state 220. However, if there are more sequences in the database, process 200 then moves to state 224 where the pointer moves to the next array in the database for comparison with the new sequence. In this way, the new sequence is aligned and compared to all sequences in the database.

Note that if the decision state 212 determines that the sequences are not homologous, the process 200 immediately moves to the decision state 218 to determine if other sequences are available for comparison in the database. Should do. Therefore, one aspect of the present invention is a computer system including a processor and a data storage device storing the nucleic acid code or the polypeptide code of the present invention. In some embodiments, the computer system further comprises a data storage device searchably stored with a reference nucleotide sequence or polypeptide sequence to be compared with the nucleic acid code or polypeptide code of the invention, and a sequence comparer to perform the comparison. Equipped with. For example, the sequence comparer may include a computer program that exhibits polymorphisms. In another aspect of this computer system, the system further comprises an identifier that identifies features of the array. A sequence comparer indicates a level of homology between compared sequences, a motif involved in a biological function of a sequence compared with the nucleic acid code of the present invention or the polypeptide code of the present invention, and a structural motif, or Structural motifs within these nucleic acid or polypeptide codes can be identified. In some embodiments, at least 2, 5, 10, 15, 20, 25, 30, 50, 75, 100, 150 or 200 of the nucleic acid code of the invention or the sequence of the polypeptide code of the invention is data stored. It may be recorded on the device. Another aspect of the invention is a method of determining the level of homology between a nucleic acid code of the invention and a reference nucleotide sequence, wherein the nucleic acid code and the reference nucleotide sequence are read by use of a computer program to determine the level of homology. And a computer program to determine the level of homology between the nucleic acid code and the reference nucleotide sequence. The computer program can be any of a number of computer program homology determinations, including those specifically described in this application, including BLAST2N with default parameters or any modified parameters. This method may be performed using the computer system described above. Also, the method of determining this level of homology reads 2, 5, 10, 15, 20, 25, 30, 50, 75, 100, 150 or 200 of the above-described nucleic acid codes of the present invention through the use of computer programs. , By determining the homology between the nucleic acid code and the reference nucleotide sequence.

FIG. 4 is a process for determining whether two sequences are homologous in a computer.
25 is a flowchart showing an embodiment of 250. The process 250 starts at a start state 252 and moves to a state 254 where the first array to compare is stored in memory. The second array to compare is then stored in memory at state 256. The process 250 then enters a state 260 where the first character of the first array is read and then a state 262 where the first character of the second array is read.
Move. It should be understood that the letters are usually A, T, C, G or U when the sequence is a nucleotide sequence. If the sequence is a protein sequence, the first and sequence sequences should be in the one-letter amino acid code for easy comparison. Next, the determination state 264 determines whether the two characters are equivalent. If they are equivalent,
Process 250 then moves to state 268 where the next character in the first and second arrays is read.
Then, it is determined whether the following characters are equivalent. If so, process 250 is 2
Repeat this operation until the two characters are not the same. If it is determined that the next two characters are not the same, process 250 moves to decision state 274 to determine if there are more characters to read from either array. If there are no more characters to read, process 250 then moves to state 276 where the level of homology between the first and second sequences is displayed to the user. Homology level is 1st
It is determined by calculating the proportion of characters that are similar between sequences in the total sequence of sequences. Therefore, the letter of the first nucleotide sequence of 100 nucleotides is changed to the second
If aligned with all the letters in the sequence, the homology level will be 100%. Alternatively, the computer program may be a computer program that compares the nucleotide sequence of the nucleic acid code of the invention with a reference nucleotide sequence to determine if the nucleic acid code of the invention differs from the reference nucleic acid sequence. Optionally,
Such programs record the length and identity of the inserted, removed or substituted nucleotides with respect to the reference polynucleotide or the sequence of the nucleic acid coding of the present invention. In one embodiment, the computer program directs the nucleotide sequence of the nucleic acid code of the invention to have one or more single nucleotide polymorphisms (SNPs) relative to the reference nucleotide sequence.
) May be included in the program. These single nucleotide polymorphisms are
Each may contain a single base substitution, insertion, or deletion. Another embodiment of this invention is the cDNA code of SEQ ID NOs: 1-297 and SEQ ID NOs: 298-5.
A method for comparing a first sequence selected from the group consisting of 94 polypeptide codes with a reference sequence, the method comprising the steps of:
Of the sequence and the reference sequence, and determining the difference between the first sequence and the reference sequence using a computer program. In some aspects of this embodiment, the step of determining the difference between the first sequence and the reference sequence comprises identifying a polymorphism.

Another aspect of the invention is a method of determining the level of homology between a polypeptide code of the invention and a reference polypeptide sequence, the method comprising the use of a computer program to determine the level of homology And reading the reference polypeptide sequence, and using a computer program to determine the level of homology between the polypeptide code and the reference polypeptide sequence. Accordingly, another aspect of the invention is a method of determining whether the nucleic acid code of the invention differs from a reference nucleotide sequence at one or more nucleotides, by the use of a computer program to identify differences between nucleic acid sequences, Reading the nucleic acid code and the reference nucleotide sequence includes identifying differences between the nucleic acid code and the reference nucleotide sequence with a computer program. In some embodiments, the computer program is a program that identifies single nucleotide polymorphisms.
This method can be implemented by the computer system described above and the method shown in FIG. Furthermore, this method uses at least 2, 5, 10 by the use of computer programs.
, 15, 20, 25, 30, 50, 75, 100, 150 or 200 of the nucleic acid code of the present invention and a reference nucleotide sequence, and the computer program identifies the difference between the nucleic acid code and the reference nucleotide sequence. May be. Therefore, another embodiment of the present invention is a method of comparing a first sequence selected from the group consisting of the nucleic acid code of the present invention or the polypeptide code of the present invention with a reference sequence, wherein the sequences are compared. Reading the first sequence and the reference sequence with a computer program that uses the computer program and determining the difference between the first sequence and the reference sequence with the computer program. In some aspects of this embodiment, the step of determining the difference between the first sequence and the reference sequence comprises identification of the polymorphism. Another aspect of the invention is a method of determining the level of identity of a first sequence selected from the group consisting of the nucleic acid code of the invention or the polypeptide code of the invention with a reference sequence, the method comprising: First, using a computer program to determine the level
And a reference sequence, and determining the level of identity between the first sequence and the reference sequence using a computer program. Other embodiments of the computer-based system may further comprise an identifier that identifies features within the nucleotide sequence of the nucleic acid code of the invention and the amino acid sequence of the polypeptide code of the invention. "Identifier"
Another embodiment of the computer-based system presents one or more programs that identify certain features within the nucleotide sequences of the nucleic acid code and the amino acid sequence of the polypeptide code of the invention described above. In one embodiment, the identifier may include a program that identifies the open reading frame of the cDNA code of the invention.

Another embodiment of the present invention is a method of identifying a feature of a sequence selected from the group consisting of the amino acid sequences of the nucleic acid or polypeptide codes of the invention, the computer program identifying the feature of the sequence. Reading the array using
A method comprising identifying a sequence characteristic using a computer program. In one aspect of this embodiment, the computer program comprises a program that identifies open reading frames. In a further embodiment, the computer program comprises a program to identify linear or structural motifs within a polypeptide sequence. FIG. 5 shows an identifier process 300 for detecting the presence of features in an array.
3 is a flow chart illustrating one embodiment of The process 300 starts at a start state 302 and then a first array to be checked for features is 115 in the memory of the computer system 100.
Move to the stored state 304. The process 300 then moves to the state 306 where the sequence feature database is opened. Such databases include a list of attributes for each feature along with the feature name. For example, the feature name may be "start codon" and the attribute may be "ATG". Another example is that the feature name is "TAATAA Box" and the feature attribute is "TAATAA". The Genetics Computer Group at the University of Wisconsin (www.gcg.com) has created an example of such a database. Once the feature database has been opened in state 306, process 300 moves to state 308 where the first feature is read from the database. At state 310, the attribute of the first feature is compared with the first array. In decision state 316 it is determined whether the feature attribute was found in the first array. If the attribute is found, then process 300 moves to state 318 where the name of the found feature is displayed to the user. The process 300 then moves to a decision state 320 to determine if more features are present in the database. If there are no further features, process 300 then ends at end state 324. However, if more features are present in the database, process 300 then reads the next sequence feature in state 326 and loops back to state 310 where the attributes of the next feature are compared against the first sequence. If the feature attribute is not found in the first array in decision state 316, process 300 proceeds directly to decision state 320 to determine if the feature is present in the database.
It should be noted that moving to.

In another embodiment, the identifier may include a molecular modeling program that can determine the conformation of the polypeptide code of the invention. Programs such as these may use any of the methods known to those of skill in the art, including methods based on homology modeling, fold recognition and abu initio methods, as described by Sternberg et al., 1999. Often, the disclosure is incorporated herein by reference in its entirety. In some embodiments, the molecular modeling program identifies the target sequence that best fits the profile representing the structural environment of the residues of the known three-dimensional protein structure. (For example, Eisenberg et al., US Pat. No. 5,436,850, July 1995.
Issued on 25th, see. The entire publication is incorporated herein by reference. 2.) In other techniques, within a given protein family, the known three-dimensional structures of several proteins are overlaid to define regions that remain structurally constant in that protein family. This protein modeling technique also uses the known conformation of homologous proteins to approximate the structure of the polypeptide code of the invention. (For example, Srinivasan, et al., US Pat. No. 5,557,53, 1996.
See Issued September 17. The entire disclosure of which is incorporated herein by reference. ) Traditional homology modeling techniques have been commonly used to build models of proteolytic enzymes and antibodies (Sowdhamini et al., (1997)).
. The comparative method can be used to develop a three-dimensional protein model even when the sequence identity between the protein of interest and the template protein is low. Proteins may fold into a similar conformation despite having low sequence identity. For example, the conformation of some helical cytokines fold into similar conformational phases despite low sequence homology. Recent developments in threading methods have allowed the identification of potential folding patterns in some situations where the structural relationship between target and template is not detectable at the sequence level. Multiple Sequence Threading
, MST) is used to perform folding recognition, and DRAGON, which is a distance geometry program, is used to infer structural equivalence from the output of threading and create a low-resolution model. Create an all-atom display using the molecular modeling package.

According to this three-step method, first, candidate templates were identified using MST, a novel folding recognition algorithm capable of threading multiple aligned sequences in one or more conformationally threading sequences. To do. In the second step, the structural similarities obtained from the MST output were translated into interresidue distance restrictions and into the distance geometry program DRAGON, along with the complementary information obtained from the prediction of secondary structure. Is entered. In this program, the constraints are combined in a bias-free manner to produce many low resolution models. In the third step, these low resolution model validations are converted to an all atom model using the molecular modeling package QUANTA and undergo energy minimization. (See, eg, Aszodi et al. (1997)). The results of the molecular modeling analysis can then be used in rational drug design techniques to identify formulations that modulate the activity of the polypeptide code of the present invention. Accordingly, another aspect of the invention is a method of identifying a feature in a nucleic acid or polypeptide code of the invention, the method comprising the step of reading the nucleic acid code and the polypeptide code by use of a computer program to identify the feature therein. Identifying the features in the nucleic acid code or the polypeptide code with a computer program. In one embodiment, the computer program comprises a program that identifies open reading frames. In a further embodiment, the computer program comprises a program to identify linear or structural motifs within a polypeptide sequence. In another embodiment, the computer program comprises a molecular modeling program. This method comprises the use of a computer program to provide a single sequence or at least 2, 5, 10, 15, 20, 25, 30, 50,
This may be done by reading 75, 100, 150 or 200 nucleic acid or polypeptide codes of the invention and identifying with a computer program features in the nucleic acid or polypeptide code. The nucleic acid code or polypeptide code of the invention can be stored and manipulated in various formats in various data processing programs. For example, these codes are M
Text in word profiles such as icrosoft Word or WordPerfect, or ASC
The II file can be stored in various database programs known to those skilled in the art, such as DB2, SYBASE, and ORACLE. In addition, numerous computer programs and databases are used as sequence data sources for sequence comparers, identifiers, or reference nucleotide sequences or polypeptide sequences to be compared with the nucleic acid code of the invention or the polypeptide code of the invention. Is possible. The following list is not meant to limit the invention, but rather to provide guidance regarding programs and databases useful for the nucleic acid code of the invention or the polypeptide code of the invention. The programs and databases you can use include, but are not limited to: MacPattern (EMBL).
, DiscoveryBase (Molecular Applications Group), GeneMine (Molecular Appl
ications Group), Look (Molecular Applications Group), MacLook (Molecular
Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altsc
hul et al., 1990), FASTA (Pearson and Lipman, 1988), FASTDB (Brutlag et al., 199).
0), Catalyst (Molecular Simulations Inc.), Catalyst / SHAPE (Molecular Sim
ulations Inc.), Cerius2.DBAccess (Molecular Simulations Inc.), HypoGen (
Molecular Simulations Inc.), Insight II, (Molecular Simulations Inc.), D
iscover (Molecular Simulations Inc.), CHARMm (Molecular Simulations Inc.
), Felix (Molecular Simulations Inc.), DelPhi, (Molecular Simulations In
c.), QuantaMM, (Molecular Simulations Inc.), Homology (Molecular Simulat
ions Inc.), Modeler (Molecular Simulations Inc.), ISIS (Molecular Simula
tions Inc.), Quanta / Protein Design (Molecular Simulations Inc.), WebLab
(Molecular Simulations Inc.), WebLab Diversity Explorer (Molecular Simul
ations Inc.), Gene Explorer (Molecular Simulations Inc.), SeqFold (Molec
ular Simulations Inc.), EMBL / Swiss protein database, MDL Available Che
micals Directory database, MDL Drug Data Report database, Comprehe
nsive Medicinal Chemistry database, Derwents's World Drug Index database, BioByteMasterFile database, Genbank database, and Genseq
n database. Many other programs and databases will be apparent to those of ordinary skill in the art in view of this disclosure.

The motifs detected using the above program include sequences encoding leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitin binding sites, alpha helices, and beta sheets, encoded proteins. It contains sequences involved in transcriptional regulation such as a signal sequence encoding a signal peptide that directs secretion, homeobox, acidic sequence, enzyme active site, substrate binding site, and enzyme cleavage site. (Conclusion) As explained above, the GENSET polynucleotides and polypeptides of the present invention, or fragments thereof, can be used for various purposes. The polynucleotide is for expressing a recombinant protein for use in analysis, characterization or therapy; the corresponding protein is preferentially expressed (either structurally or at a particular stage of tissue differentiation or development, Or as a marker for a tissue to be treated (in a disease state); as a molecular weight marker on Southern gels; as a chromosomal marker or tag (if labeled) to identify a chromosome or map the location of related genes; As reagents of an assay (including labeling reagents) designed to quantitatively measure GENSET expression levels in; to identify potential genetic disorders compared to endogenous DNA sequences in patients; novel As a probe for hybridizing and therefore discovering related DNA sequences of; PCR for genetic fingerprinting
As a source of information for inducing primers; to select and create oligomers for attachment to “gene chips” or other supports, including examination of expression patterns; To produce; and as an antigen to produce anti-DNA antibodies or to elicit other immune responses. If the polynucleotide encodes a protein that binds, or potentially binds, other proteins (eg, in receptor-ligand interactions, etc.), the polynucleotide is further described in an interaction trap assay (eg, Gyuris
Et al., (1993)), to identify polynucleotides encoding other proteins that undergo binding thereto, or to identify inhibitors of binding interactions. . The proteins or polypeptides provided by the invention also include panels of multiple proteins for high throughput screening, in assays for measuring biological activity; to elicit antibodies or other immune responses; As reagents (including labeling reagents) in assays designed to quantitatively measure levels of proteins (or receptors) in body fluids; tissues in which the corresponding proteins are preferentially expressed ( It can be used structurally or as a marker (at a particular stage of tissue differentiation or development, or in a disease state); and, of course, to isolate correlating receptors or ligands. If a protein binds, or potentially binds (encodes a protein) to another protein (eg, in a receptor-ligand interaction), the protein may be associated with another protein that causes it to bind. It can be used for identification or for identifying inhibitors of binding interactions. The proteins involved in these binding interactions can also be used to screen peptides or small molecule inhibitors or agonists of binding interactions.

All of these research utilities can be developed in reagent grade or kit formats that can be sold as research products. Methods for performing the above-described uses are known to those skilled in the art. References disclosing such methods include, but are not limited to, the following: "Molecular Clon
ing; A Laboratory Manual ", 2d ed., Cole Spring Harbor Laboratory Press,
Sambrook, J., EF Fritsch and T. Maniatis eds. 1989, and Methods
in Enzymology; Guide to Molecular Cloning Techniques, '' Academic Press
Berger and Kimmel eds. 1987, the entire contents of these disclosures are incorporated herein by reference. The polynucleotides and proteins of the present invention can also be used as a nutritional source or a nutritional supplement source. Applications such as these include, but are not limited to, applications as protein or amino acid supplements, carbon sources, nitrogen sources and carbohydrate sources. In these cases, the protein or polynucleotide of the invention may be added to the diet of a particular organism, or in the form of powder, tablets, solutions, suspensions or capsules, apart from the diet, in solid or liquid preparations. It can be administered as a product. In the case of microorganisms, the protein or polynucleotide of the present invention can be added to or in the medium in which the microorganisms are cultured. Although the present invention has been described in particular preferred embodiments, other embodiments that will be apparent to those of ordinary skill in the art in view of the disclosure of this application are also contemplated by the claims of the present invention. Accordingly, the claims of the present invention are defined solely by reference to the appended claims.

[0483]

【Example】

Preparation of Antibody Compositions against GENSET Protein Relatively pure proteins or polypeptides are isolated from transfected or transformed cells containing an expression vector encoding the GENSET protein or a portion thereof. The concentration of protein in the final preparation is adjusted to a level of a few μg / ml, for example by concentration with an Amicon filter device. Monoclonal or polyclonal antibodies to the protein can then be prepared as follows: <A. Production of Monoclonal Antibodies by Hybridoma Fusion> Monoclonal antibodies to epitopes in the GENSET protein or part thereof can be obtained from mouse hybridomas by Kohler and Milstein. (1975) or a derivative method thereof. Harlow and Lane
See also (1988). That is, mice are repeatedly inoculated with several micrograms of GENSET protein or a portion thereof over a period of several weeks. The mouse is then sacrificed and antibody-producing spleen cells are isolated. Spleen cells were fused with mouse myeloma cells using polyethylene glycol, and excess unfused cells were killed by growing the system on selective medium containing aminopterin (HAT medium). The successfully fused fused cells were diluted and an aliquot of the dilution was placed in the wells of a microtiter plate where the culture continued to grow. A clone producing an antibody can be identified by detecting the antibody in the supernatant of the well by an immunoassay procedure such as ELISA or a derivative method thereof. The assay procedure was first described by Engvall, (1980), the entire disclosure of which is incorporated herein by reference. Selected positive clones grow and their monoclonal antibody product can be collected and used. Detailed procedures for producing monoclonal antibodies are described in Davis et al. (1986) 21-2.
It is described in the section. <B. Production of Polyclonal Antibody by Immunization> A polyclonal antiserum containing an antibody against a GENSET protein or a heterogeneous epitope thereof is unmodified or modified to enhance immunogenicity.
It can be prepared by immunizing a suitable non-human animal with the ET protein or a portion thereof. A suitable non-human animal is a non-human mammal,
Preferably, mouse, rat, rabbit, goat, or horse is usually selected. Also,
The crude preparation enriched in GENSET concentration can be used to produce antibodies. Such proteins, fragments or preparations are introduced into a non-human mammal in the presence of a suitable adjuvant known in the art (eg aluminum hydroxide, RIBI etc.). Furthermore, the protein, fragment or preparation may be pre-treated with a preparation which increases the antigenicity, such preparations are known in the art, eg methylated bovine serum albumin (mBSA), bovine serum. Albumin (BSA)
, Hepatitis B surface antigen and keyhole limpet hemocyanin (KLH). Sera of immunized animals are collected, treated and tested according to known procedures. If the serum contains polyclonal antibodies to unwanted epitopes, the polyclonal antibodies can be purified by immunoaffinity chromatography.

Efficient polyclonal antibody production is influenced by many factors related to both antigen and host type. Also, different host animals have different responses to the site of inoculation and dose, resulting in low titer antisera whether the dose of antigen is inadequate or excessive. Small doses (ng levels) of challenge at multiple intradermal sites appear to be the most reliable. Techniques for producing and processing polyclonal antisera are known in the art. An effective immunization protocol in rabbits is Va
Itukaitis et al. (1971), the disclosure of which is incorporated herein by reference in its entirety. Booster injections can be given periodically and antisera can be collected at a time when the antibody titer begins to drop to known antigen concentrations, for example by semi-quantitative measurement by double immunodiffusion in agar. See, for example, Ouchterlony et al. (1973). The entire publication is incorporated herein by reference. Plateau concentration of antibody is usually in the range of 0.1-0.2 mg / ml of serum (about 12 uM). The affinity of the antiserum for the antigen is determined by constructing a competitive binding curve. This is
Written by Fisher (1980), the disclosure of which is incorporated herein by reference in its entirety. Antibody preparations prepared according to either monoclonal or polyclonal protocol procedures are useful in quantitative immunoassays to determine the concentration of antigen-bearing substances in biological samples. They are also used semi-quantitatively or qualitatively to confirm the presence of antigens in biological samples. These antibodies can be used in therapeutic compositions to kill protein-expressing cells or reduce the levels of proteins in the body. Biological Assay <Assay for GENSET Secreted Protein to Determine Whether GENSET Secreted Protein Binds to Cell Surface> Secreted protein encoded by GENSET cDNA, preferably SEQ ID NO: 242
The -272 and 274-384 proteins, or fragments thereof, are cloned into an expression vector. Proteins are purified by size, charge, immunochromatography, and other techniques known to those of skill in the art. After purification, the protein is labeled using techniques known to those of skill in the art. The labeled protein is incubated with cells or cell lines derived from various organs or tissues to bind the protein to any receptor present on the cell surface. After incubation, cells are washed to remove non-specifically bound proteins. Labeled protein is detected by autoradiography. Alternatively, the unlabeled protein can be incubated with cells and detected with an antibody having a label detectable by binding to a fluorescent molecule or the like.

The specificity of binding to the cell surface can be analyzed by a competition assay in which different amounts of unlabeled protein are incubated with the labeled protein. The amount of labeled protein bound to the cell surface decreases as the amount of unlabeled protein in competition with the labeled protein increases. As a control, different amounts of unlabeled protein unrelated to labeled protein were included in some binding reactions. The amount of labeled protein bound to the cell surface did not decrease in the binding reaction when the amount of irrelevant unlabeled protein was increased, indicating that the protein encoded by the cDNA specifically bound to the cell surface. Show. As described herein, it has become clear that secreted proteins have several important physiological effects and, as a result, are valuable therapeutic resources. Secreted proteins encoded by the cDNAs or fragments thereof produced by any of the methods described herein can be evaluated as described below to determine their physiological activity. <Assay for Cytokine, Cell Proliferation or Cell Differentiation Activity of GENSET Protein or Fragment Thereof> Secreted protein may act as a cytokine or may affect cell proliferation or differentiation. Many protein factors discovered to date, including all known cytokines, exhibit activity in one or more factor-dependent cell proliferation assays, thus the assay serves as a convenient confirmation of cytokine activity. The activity of the protein of the present invention is 32D, DA2, DA1G, T10, B9, B9 / 11, BaF3.
, MC9 / G, M + (preB M +), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7
Clarified by several common factor-dependent cell proliferation assays in cell lines, including but not limited to c and CMK. The protein encoded by the cDNA of the present invention or a fragment thereof,
Their ability to control T cell, or thymocyte proliferation, can be assessed by assays as described in the references above or below, which are incorporated herein by reference. Yes: Current Protocols in Immunology, Ed. B
y JE Coligan et al., Greene Publishing Associates and Wiley-Interscience;
Takai et al., J. Immunol. 137: 3494-3500, 1986. Bertagnolli et al., J. Immunol. 14
5: 1706-1712, 1990. Bertagnolli et al., Cellular Immunology 133: 327-341, 1991.
Bertagnolli, et al., J. Immunol. 149: 3778-3783, 1992; Bowman et al., J. Immunol.
152: 1756-1761, 1994.

In addition, numerous assays are known for cytokine production and / or proliferation of spleen cells, lymph node cells and thymocytes. These techniques are
Including those published in: Current Protocols in Immunology. J
.E. Coligan et al., Eds., Vol 1 pp. 3.12.1-3.12.14 John Wiley and Sons, Toro
nto. 1994; and Schreiber, RD Current Protocols in Immunology., supra.
Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994. The protein encoded by the cDNA of the present invention has the ability to control proliferation and differentiation of hematopoietic or lymphopoietic cells. You can also test. Many such assays for activity are known to those of skill in the art, including those in the following references and are incorporated herein by reference: Bottomly, K., Da.
vis, LS and Lipsky, PE, Measurement of Human and Murine Interleuki
n 2 and Interleukin 4, Current Protocols in Immunology., JE Coligan et al., Eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVr.
ies et al., J. Exp. Med. 173: 1205-1211, 1991; Moreau et al., Nature 36: 690-692, 19
88; Greenberger et al., Proc. Natl. Acad. Sci. USA 80: 2931-2938, 1983; Nor.
dan, R., Measurement of Mouse and Human Interleukin 6 Current Protocols
in Immunology. JE Coligan et al., Eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley a
nd Sons, Toronto. 1991; Smith et al., Proc. Natl. Acad. Sci. USA 83: 1857-1.
861, 1986; Bennett, F., Giannotti, J., Clark, SC and Turner, KJ, Me.
asurement of Human Interleukin 11 Current Protocols in Immunology. JE
Coligan et al., Eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Ci
arletta, A., Giannotti, J., Clark, SC and Turner, KJ, Measurement
of Mouse and Human Interleukin 9 Current Protocols in Immunology. JE C
oligan et al., Eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991. The proteins encoded by the cDNAs of the present invention may also be assayed for their ability to control the response of T cells to antigens. it can. Many such assays for activity are known to those of skill in the art, including the assays of the references described below, and are incorporated herein by reference: Current Protocols in
Immunology Chapter 3 (In vitro Assays for Mouse Lymphocyte Function), Chapter 6
Chapter (Cytokines and Their Cellular Receptors) and Chapter 7 (Immunologic Stu)
dies in Humans), JE Coligan et al., Eds. Greene Publishing Associates and
Wiley-Interscience; Weinberger et al., Proc. Natl. Acad. Sci. USA 77: 6091-60.
95, 1980; Weinberger et al., Eur. J. Immun. 11: 405-411, 1981; Takai et al., J. Im.
munol. 137: 3494-3500, 1986; Takai et al., J. Immunol. 140: 508-512, 1988.

[0487] Cytokines, proteins that exhibit cell proliferation or cell differentiation activity can then be formulated as pharmaceutical agents and used to treat clinical conditions in which induction of cell proliferation or differentiation is beneficial. Alternatively, as described in more detail below, the genes encoding these proteins, or the nucleic acids that control the expression of these proteins may be introduced into appropriate host cells to increase or decrease protein expression as desired. it can. <Assay for Activity of GENSET Protein or Fragment thereof as Immune System Regulator> The protein encoded by the cDNA of the present invention can be evaluated for its effect as an immune regulator. For example, the proteins can be evaluated for their activity in affecting cytotoxicity of their thymocytes or splenocytes. Many such assays for activity are known to those of skill in the art, including those in the references described below, and are incorporated herein by reference: Current Protocols in Immunology Chapter 3 (In vitro Assays for Mouse Ly
mphocyte Function 3.1-3.19) and Chapter 7 (Immunologic Studies in Humans)
), JE Coligan et al., Eds. Greene Publishing Associates and Wiley-Intersc
ience; Herrmann et al., Proc. Natl. Acad. Sci. USA 78: 2488-2492, 1981; Herrma.
nn et al., J. Immunol. 128: 1968-1974, 1982; Handa et al., J. Immunol. 135: 1564-15.
72, 1985; Takai et al., J. Immunol. 137: 3494-3500, 1986; Takai et al., J. Immuno.
l. 140: 508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78: 2488-24.
92, 1981; Herrmann et al., J. Immunol. 128: 1968-1974, 1982; Handa et al., J. Immun.
ol. 135: 1564-1572, 1985; Takai et al., J. Immunol. 137: 3494-3500, 1986; Bowma.
n et al., J. Virology 61: 1992-1998; Takai et al., J. Immunol. 140: 508-512, 1988; B.
ertagnolli et al., Cellular Immunology 133: 327-341, 1991; Brown et al., J. Immunol.
. 153: 3079-3092, 1994. The proteins encoded by the cDNAs of the invention can also be evaluated for their effect on T cell-dependent immunoglobulin responses and isotype switching. Many assays for such activity, including assays published in the following references, are known to those of skill in the art and are incorporated herein by reference: Bottomly, K. , Davis, LS Mond, JJ and Br
unswick, M Current Protocols in Immunology Assays for B Cell Function:
In vitro Antibody Production, Vol 1 pp. 3.8.1-3.8.16. JE Coligan et al Eds
., John Wiley and Sons, Toronto. 1994.

The protein encoded by the cDNA of the present invention can be evaluated for its effect on immune effector cells, including the effect on Th1 cells and cytotoxic lymphocytes. Many such assays for activity are known to those of skill in the art, including the assays of the references described below and are incorporated herein by reference: Current Protocols in Immunology Chapter 3 (In vitro Assay
s for Mouse Lymphocyte Function 3.1 to 3.19) and Chapter 7 (Immunologic Stud)
ies in Humans), JE Coligan et al., Eds. Greene Publishing Associates and
Wiley-Interscience; Takai et al., J. Immunol. 137: 3494-3500, 1986; Takai et al.
J. Immunol. 140: 508-512, 1988; Bertagnolli et al., J. Immunol. 149: 3778-3783.
, 1992. The proteins encoded by the cDNAs of the present invention can also be evaluated for their effect on dendritic cell-mediated activation of undifferentiated T (Th0) cells. Many such assays for activity are known to those of skill in the art, including those published in the following references, which are incorporated herein by reference: Guery et al., J. Immunol. 134: 536-544, 1995; Inaba et al., Journal of Exper.
imental Medicine 173: 549-559, 1991; Macatonia et al., Journal of Immunology 1
54: 5071-5079, 1995; Journal of Experimental Medicine 182: 255-260, 1995;
Nair et al., Journal of Virology 67: 4062-4069, 1993; Huang et al., Science 264: 96.
1-965, 1994; Macatonia et al., Journal of Experimental Medicine 169: 1255-1264.
, 1989; Bhardwaj et al., Journal of Clinical Investigation 94: 797-807, 1994;
And Inaba et al., Journal of Experimental Medicine 172: 631-640, 1990. The proteins encoded by the cDNAs of the present invention can also be evaluated for their effect on lymphocyte longevity. Many assays for such activity, including those published in the following references, are known to those of skill in the art and are incorporated herein by reference: Darzynkiewicz et al., Cytomet.
ry 13: 795-808, 1992; Gorczyca et al., Leukemia 7: 659-670, 1993; Gorczyca et al., C.
ancer Research 53: 1945-1951, 1993; Itoh et al., Cell 66: 233-243, 1991; Zachar.
chuk, Journal of Immunology 145: 4037-4045, 1990; Zamai et al., Cytometry 14: 8.
91-897, 1993; Gorczyca et al., International Journal of Oncology 1: 639-648, 1
992.

Assays for proteins that affect T cell involvement, and early stages of development, include:
Including but not limited to those described below: Anti
ca et al., Blood 84: 111-117, 1994; Fine et al., Cellular immunology 155: 111-122, 1
994; Galy et al., Blood 85: 2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA.
88: 7548-7551, 1991. Then, a protein that exhibits activity as an activity of an immune system regulator can be formulated into a drug and used to treat clinical conditions in which regulation of immune activity is beneficial. For example, the protein may contribute to T and / or B lymphocyte growth and proliferation (
Up or down) regulation, and may be useful in the treatment of various immune deficiencies and disorders, including cytolytic activity effects of NK cells and other cell populations, including severe deciphered immunodeficiency (SICD). These immunodeficiencies can result from genetic or viral (eg HIV) as well as bacterial or fungal infections, or as a result of autoimmune disorders. More specifically, infectious diseases caused by viral, bacterial, fungal or other infections can affect a variety of diseases including HIV, hepatitis virus, herpes virus, mycobacteria, Leishmania, malaria parasites, and candidiasis. It includes fungal infections and can be treated with the proteins of the invention. Of course, in this respect, the proteins of the invention may also be useful when it is generally desirable to boost the immune system, ie in treating cancer. Autoimmune disorders treatable by the proteins of the invention include, for example, connective tissue disease,
Multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pneumonia,
Includes Guillain-Barre syndrome, autoimmune thyroiditis, insulin-dependent diabetes mellitus, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such proteins of the invention may be useful in the treatment of allergic reactions and medical conditions such as asthma (especially allergic asthma) or respiratory problems. Other medical conditions where immunosuppression is desired (including, for example, organ transplantation) can also be treated with the proteins of the invention. It may also be possible to control the immune response in several ways using the proteins of the invention. Down-regulation can also take the form of suppressing or blocking an already ongoing immune response or preventing the induction of an immune response. The function of activated T cells can be suppressed by suppressing the T cell response, or by inducing specific resistance to the T cell, or both. Immunosuppression of T cell responses is generally an active and antigen-nonspecific process that requires constant exposure of T cells to suppressors. Resistance associated with T cell unresponsiveness or induction of anergy is generally immunospecific because it is antigen specific and persists after termination of exposure to tolerizing agents. It is distinct from suppression. In operation, tolerance was re-exposed to specific antigen in the absence of tolerizing agent,
It can be demonstrated by the lack of a T cell response.

Downregulating or preventing one or more antigenic functions, including but not limited to B lymphocyte antigenic functions (eg, B7, etc.), eg, high level lymphokine synthesis by activated T cells. Prevention is useful in tissue, skin and organ transplants and in the context of graft-versus-host disease (GVHD). For example, blocking T cell function results in reduced tissue destruction in tissue transplants. Typically, rejection of grafts in tissue transplants results from their foreign body being recognized by T cells,
It is followed by an immune reaction that destroys the graft. Molecules that suppress or block the interaction of B7 lymphocyte antigens with their natural ligands on immune cells (soluble monomeric peptides, alone or in monomeric form of other B lymphocyte antigens. If an active peptide (for example, B7-1, B7-3) or a B7-2 activity in combination with a blocking antibody is administered before transplantation, the corresponding costimulatory signal will not be transmitted, It is possible to reach the binding of the molecule with the natural ligand on the immune cell. Blocking the B lymphocyte antigen function here interferes with cytokine synthesis by immune cells such as T cells and thus functions as an immunosuppressant. Furthermore, the lack of costimulation is sufficient to anergize T cells, thereby inducing tolerance in transplant recipients. Inducing long-term tolerance with B lymphocyte antigen blocking reagents may circumvent the need for repeated administration of these blocking reagents. To achieve sufficient immunosuppression or resistance in transplant recipients, it may also be necessary to block the function of the B lymphocyte antigen combination. The efficacy of certain blocking agents in preventing organ transplant rejection or GVHD can be evaluated using animal models with predictable efficacy in humans. Examples of suitable organ systems that can be used include allogeneic heart transplants in rats and xenogeneic Langerhans islet cell transplants in mice, both Lenschow et al., Science 257: 789-792 (1992) and Turka et al. , Proc. Acad. Sci USA, 89: 11102-11105 (1992), to examine the in vivo immunosuppressive effect of the CTLA4Ig fusion protein. In addition, GVHD mouse model (Paul ed., Fundamental
Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to measure the effect of blocking B lymphocyte antigen function on disease progression in vivo.

In addition, blocking antigen function may be therapeutically useful in treating autoimmune diseases. Many autoimmune disorders result from improper activation of T cells that are reactive to self tissue and promote the production of cytokines and autoantibodies involved in the pathology of the disease. By preventing activation of autoreactive T cells, symptoms of the disease can be reduced or eliminated. Administration of a reagent that blocks T-cell costimulation by blocking the interaction between the receptor and the ligand for the B lymphocyte antigen appears to be involved in the disease process by inhibiting T-cell activation. It can be used to prevent the production of autoantibodies or cytokines derived from T cells. In addition, blocking reagents may induce antigen-specific tolerance of autoreactive T cells, reducing disease over time. The efficacy of blocking agents in preventing or reducing autoimmune disorders can be determined by the use of several well characterized animal models of human autoimmune disease. Examples include murine experimental autoimmune encephalitis, systemic lupus erythematosus in MRL / pr / pr or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes in OD and BB rats, and murine experimental myasthenia gravis. (Paul ed., Fundamental Immunology, Raven Press, New Y
ork, 1989, pp. 840-856). Antigen function (preferably B lymphocyte antigen function) as an up-regulator of immune response
Up-regulation of can also be useful in therapy. Upregulation of the immune response may take the form of enhancing the existing immune response or eliciting an early immune response. For example, stimulating B lymphocyte antigen function to enhance the immune response may be useful against viral infections. Furthermore, systemic viral diseases such as influenza, common cold, and encephalitis can be alleviated by systemic administration of stimulatory forms of B lymphocyte antigens. Alternatively, the T cells were removed from the patient, the T cells were stimulated with APC to which a viral antigen was applied, which was expressed only with the peptide of the present invention or with the soluble peptide of the present invention, and activated in vitro. The T cells can be reintroduced into the patient described above to enhance the antiviral properties of infected patients. This allows infected cells to transmit costimulatory signals to T cells in vivo,
Thereby, T cells can be activated.

In other applications, upregulation or enhancement of antigen function (preferably B lymphocyte antigen function) may be useful in inducing tumor immunity. Tumor-specific tumor cells of a subject are administered by administering to the subject tumor cells (for example, sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) into which a nucleic acid encoding at least one of the peptides of the present invention has been introduced. Resistance can be overcome. If desired, tumor cells may be transgenic to express a combination of peptides. For example, a tumor cell obtained from a patient is treated with an expression vector that directs the expression of a peptide having only B-7-2 like activity or B-7-1 like activity and / or B-7-3 like activity. , Can be transduced in vitro. The transgenic tumor cells are returned to the patient described above and the peptide is expressed on the transgenic cell surface. Alternatively, gene therapy techniques can be used to target tumor cells for gene transfer in vivo. The presence of the peptides of the invention having the activity of B lymphocyte antigens on the surface of tumor cells is
It provides T cells with the co-stimulatory signals necessary to induce a T cell-mediated immune response against transgenic tumor cells. In addition, tumor cells deficient in MHC class I or MHC class II molecules or unable to re-express sufficient amounts of MHC class I or MHC class II molecules are treated with MHC class I α chain proteins and β2 microglobulin proteins, or MHC class I Full-length or fragments of class II α-chain proteins and MHC class II β-chain proteins (eg, a fragment with a truncated cytoplasmic domain)
Can be transfected with a nucleic acid that encodes MHC class I or
MHC class II proteins can be expressed on the surface of cells. Expression of appropriate class I or class II MHC and B lymphocyte antigens (eg B7-1, B7-2, B7-3)
In combination with a peptide having the activity of eliciting a T cell-mediated immune response against transgenic tumor cells. If desired, MHC class I such as invariant chain
A gene encoding an antisense construct that blocks the expression of I-related proteins
It can also be co-transfected with DNA encoding a peptide having the activity of lymphocyte antigens to promote the presentation of tumor-associated antigens and the induction of tumor-specific immunity. Therefore, induction of a T cell-mediated immune response in humans may be sufficient to overcome tumor-specific resistance in patients. Alternatively, as further detailed below, the genes encoding these proteins, or the nucleic acids that control the expression of these proteins are introduced into appropriate host cells to optionally increase or decrease the expression of the proteins. You can also

<Assay for Hematopoiesis-Regulating Activity of GENSET Protein or Fragment Thereof> The protein encoded by the cDNA of the present invention or a fragment thereof can also be evaluated for their hematopoiesis-regulating activity. For example, the effect of the protein on the differentiation of embryonic stem cells can be evaluated. Many such assays for activity are known to those of skill in the art, including those published in the following references and are incorporated herein by reference: Johansson et al., Ce.
llular Biology 15: 141-151, 1995; Keller et al., Molecular and Cellular Biolog.
y 13: 473-486, 1993; McClanahan et al., Blood 81: 2903-2915, 1993. The proteins encoded by the cDNAs or fragments thereof of the present invention can also be evaluated for their effect on stem cell longevity and stem cell differentiation. Many assays for such activity, including assays published in the following references, are known to those of skill in the art and are incorporated herein by reference: Freshney, MG Culture. Methylcellu of of Hematopoietic Cells
lose Colony Forming Assays. RI Freshney, et al., Eds. pp. 265-268, Wiley-L.
iss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89
: 5907-5911, 1992; McNiece, IK and Briddell, RA Culture of Hematopo
ietic Cells Primitive Hematopoietic Colony Forming Cells with High Prol
iferative Potential. RI Freshney, et al., eds. Vol pp. 23-39, Wiley-Liss,
Inc., New York, NY. 1994; Neben et al., Experimental Hematology 22: 353-359, 1
994; Ploemacher, RE Culture of Hematopoietic Cells Cobblestone Area F
orming Cell Assay. RI Freshney, et al., Eds. pp. 1-21, Wiley-Liss, Inc., N
ew York, NY. 1994; Spooncer, E., Dexter, M. and Allen, T. Culture of
Hematopoietic Cells Long Term Bone Marrow Cultures in the Presence of S
tromal Cells. RI Freshney, et al., Eds. pp. 163-179, Wiley-Liss, Inc., New.
York, NY. 1994; and Sutherland, HJ Culture of Hematopoietic Cells
Long Term Culture Initiating Cell Assay. RI Freshney, et al., Eds. Pp. 139.
-162, Wiley-Liss, Inc., New York, NY. 1994. The protein exhibiting hematopoiesis-regulating activity can then be formulated as a drug and used to treat clinical conditions in which hematopoiesis control is beneficial. For example, the proteins of the invention may be useful in controlling hematopoiesis, and thus treating defects in myeloid or lymphoid cells. We show that hematopoietic regulation is involved in the minimal biological activity that supports colony forming cells or factor-dependent cell lines. Ie, involved in supporting growth and proliferation of erythroid progenitor cells, alone or in combination with other cytokines, thereby treating, for example, various anemias or stimulating erythroid precursor and / or erythroid cell production. Have utility in combination with radiation / chemotherapy; eg, bone marrow-like cells such as granulocytes and monocytes / macrophages that are useful in combination with chemotherapy to prevent or treat severe myelosuppression Involved in supporting the growth and proliferation of (i.e., conventional CSF activity); megakaryocytes,
It is thus involved in supporting the growth and proliferation of platelets, thereby preventing or treating various platelet disorders such as thrombocytopenia, and in general can be used as a surrogate for platelet transfusion or as a complement thereto; and / or any of the above. Or involved in supporting the growth and proliferation of hematopoietic stem cells capable of maturing into all hematopoietic cells, and thus a variety of stem cell disorders, including, but not limited to, aplastic anemia and paroxysmal nocturnal hemoglobinuria, usually Establish therapeutic utility for those treated by transplantation, as well as post-irradiation / chemotherapy stem cell compartment in vivo or in vitro (ie, combined with bone marrow transplant or peripheral progenitor cell transplant (allogeneic or xenogeneic)) ) Involved in supporting repopulation as normal cells or cells genetically engineered for gene therapy.

Alternatively, as described in further detail below, the genes encoding these proteins, or the nucleic acids that control the expression of these proteins are introduced into a suitable host cell to increase expression of the proteins as desired. Or it can be reduced. <Assay for Control of Tissue Growth by GENSET Protein or Fragment thereof> The protein encoded by the cDNA of the present invention or a fragment thereof can also be evaluated for their effect on tissue growth. Many such assays for activity are described in International Patent Publication No. WO95 / 16035, International Patent Publication No. WO9.
It is known to the person skilled in the art, including the assay method published in 5/05846 and International Patent Publication No. WO 91/07491, and is incorporated herein by reference. Assays for wound healing activity are described by Eaglelstein and Mertz, J. Invest. Derm.
Winter, Epidermal Wound Healing, modified by atol 71: 382-84 (1978)
pps. 71-112 (Maibach, H1 and Rovee, DT, eds.), Year Book Medical Pub
Including but not limited to those described in lishers, Inc., Chicago, which is incorporated herein by reference. The proteins involved in controlling tissue growth can then be prepared as pharmaceuticals and used to treat clinical conditions in which controlling tissue growth would be beneficial. For example, the proteins of the invention are useful in compositions used for the growth or regeneration of bone, cartilage, tendons, ligaments and / or nerve tissue, as well as wound healing and tissue repair and replacement, as well as treatment of burns, incisions and ulcers. Can be. The proteins of the present invention that induce cartilage and / or bone growth in environments where bone is not normally formed have application in humans and other animals for fractures and cartilage damage or healing of cartilage damage. Thus, preparations using the proteins of the invention may have prophylactic use in reducing closure and open fractures, as well as improving immobilization of artificial joints. New bone formation induced by osteogenic agents contributes to the repair of congenital, trauma-induced or cancer resection-induced craniofacial defects and is also useful in cosmetic plastic surgery. Furthermore, the proteins of the present invention can be used in the treatment of periodontal disease and other tooth repair processes. Such formulations may provide an environment that attracts osteogenic cells, stimulates osteogenic cell growth, or induces differentiation of osteogenic cell precursors. Furthermore, the protein of the present invention promotes the repair of bone and / or cartilage, or the process of tissue destruction (collagenase activity,
It is useful in treating osteoporosis or osteoarthritis by blocking osteoclast activity, etc.).

Another category of tissue regeneration activity that may be attributed to the proteins of the present invention is tendon / ligament formation. Proteins of the invention that induce tendon / ligament-like tissue, or other tissue formed in an environment where such tissue would not normally form, are found in tendons or ligaments lacerations, deformities and in other humans and other animals. It can be applied to heal defects of tendons or ligaments. Preparations using such tendon / ligament-like tissue-inducing proteins provide prophylactic applications to prevent damage to tendon or ligament tissue, as well as improved fixation of the tendon or ligament to bone or other tissue, and , There may be applications to repair defects in tendon or ligament tissue. The novel tendon / ligamentary tissue induced by the composition of the present invention contributes to the repair of other tendon or ligamentous defects that are congenital, trauma-induced or otherwise, and, in cosmetic plastic surgery, tendon or ligamentous tissue. It is useful for attaching or repairing The composition of the present invention attracts tendon-forming cells or ligament-forming cells, promotes the growth of tendon-forming cells or ligament-forming cells, induces differentiation of tendon-forming cells or precursors of ligament-forming cells, or in vivo. An environment may be provided to induce the growth of tendon / ligament cells or progenitors in vitro to bring back tissue repair. Additionally, the compositions of the present invention may be useful in treating tendinitis, carpal tunnels and other tendon or ligament defects. The composition can also include a suitable substrate and / or separating agent as a carrier, as is known in the art. In addition, the protein of the present invention, the proliferation of nerve cells and regeneration of nerve and brain tissue,
That is, it may also be useful in the treatment of central nervous system and peripheral nervous system diseases and neuropathy, as well as in mechanical and traumatic disorders involving degeneration, death or trauma of nerve cells or tissue. More specifically, the protein is associated with diseases of the peripheral nervous system such as peripheral nerve injury, peripheral neuropathy and localized neuropathy, and Alzheimer's,
It can be used to treat diseases of the central nervous system such as Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. further,
Medical conditions that can be treated in accordance with the present invention include spinal cord disorders, mechanical and traumatic disorders such as head trauma, and cerebrovascular diseases such as stroke. Peripheral neuropathy resulting from chemotherapy or other medical treatment may be treatable with the proteins of the invention. The proteins of the invention are useful in better, rapid closure of non-healing wounds including, but not limited to, pressure ulcers, vascular insufficiency-related ulcers, surgical and traumatic wounds and the like.

The protein of the present invention includes organs (eg, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth muscle, skeletal muscle or myocardium) and vascular (including vascular endothelium).
It is expected to exhibit the activity of producing or regenerating other tissues, such as tissues, or promoting the growth of cells, including such tissues. Some of the desired effects may be inhibition or modulation of fibrotic scars, which allows normal tissue to produce. The proteins of the invention may exhibit angiogenic activity. In addition, the proteins of the invention may be useful in the protection or regeneration of the intestine and in the treatment of pathologies resulting from lung and liver fibrosis, reperfusion injury in various tissues, and systemic cytokine disorders. Furthermore, the protein of the present invention may be useful in promoting or suppressing differentiation of the above-mentioned tissue from progenitor tissue or cells, or in suppressing growth of the above-mentioned tissue. Alternatively, as described in further detail below, introducing genes encoding these proteins, or nucleic acids controlling the expression of these proteins into a suitable host cell to optionally increase or decrease expression of the proteins. You can also <Assay for control of reproductive hormone or cell motility of GENSET protein or fragment thereof> The protein encoded by the cDNA of the present invention or a fragment thereof can also be evaluated for its ability to control reproductive hormone such as follicle stimulating hormone. Many assays for such activity, including those published in the following references, are known to those of skill in the art and are incorporated herein by reference: Vale et al., Endocrinology. 91: 562-572, 1972; Ling et al., Nature 321.
: 779-782, 1986; Vale et al., Nature 321: 776-779, 1986; Mason et al., Nature 318: 65.
9-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83: 3091-3095, 1986. Cu.
rrent Protocols in Immunology Chapter 6.12 (Measurement of Alpha and Beta Che
mokines), JE Coligan et al., Eds. Greene Publishing Associates and Wiley-
Intersciece; Taub et al., J. Clin. Invest. 95: 1370-1376, 1995; Lind et al., APM.
IS 103: 140-146, 1995; Muller et al., Eur. J. Immunol. 25: 1744-1748; Gruber et al., J. of Immunol. 152: 5860-5867, 1994; Johnston et al., J. of Immunol. 153:
1762-1768, 1994.

Proteins that are active as reproductive hormone or cell motility regulators can then be formulated as pharmaceuticals and used to treat clinical conditions in which reproductive hormone or cell motility control is beneficial. For example, the proteins of the invention may exhibit activin-related or inhibin-related activity. The characteristics of Inhibin are:
The ability of activin to inhibit the release of follicle-stimulating hormone (FSH)
The ability to stimulate the release of follicle-stimulating hormone (FSH). Thus, the proteins of the invention are based on the ability of inhibin to reduce fertility in female mammals and reduce spermatogenesis in male mammals, either alone or as a heterodimer with a member of a protein containing the inhibin α subunit. It may be useful as a contraceptive. Administration of other inhibins in sufficient amounts induces infertility in these mammals. Alternatively, the protein of the invention stimulates FSH release from cells of the anterior pituitary as a homodimer of the protein or a heterodimer of inhibin-B with a protein subunit distinct from the protein. It may be useful as a fertility-inducing therapeutic agent based on the ability of the activin molecule. See, for example, U.S. Pat. No. 4,798,885. The entire publication is incorporated herein by reference. Furthermore, the proteins of the invention may be useful in accelerating the emergence of fertility in sexually immature mammals to increase the lifelong fertility of livestock such as cows, sheep and pigs. Alternatively, as described in further detail below, introducing genes encoding these proteins, or nucleic acids controlling the expression of these proteins into a suitable host cell to optionally increase or decrease expression of the proteins. You can also <Assay for chemotaxis / chemokinesis activity of GENSET protein or fragment thereof> The protein encoded by the cDNA of the present invention or its fragment can also be evaluated for their chemotaxis / chemokinesis activity. For example, the proteins of the invention have chemotactic or chemokinetic activity on mammalian cells including, for example, monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial cells and / or endothelial cells. (Eg acting as a chemokine). The chemotactic and chemokinesis proteins can be used to move or attract the desired cell population to the desired site of action. Chemotaxis or chemokinesis proteins offer certain advantages in the treatment of wounds and other tissue trauma, as well as localized infections. For example, the attraction of lymphocytes, monocytes or neutrophils to the tumor or site of infection will result in an improved immune response to the tumor or infectious agent.

A protein or peptide has chemotactic activity for a particular cell population due to:
In cases where it is possible, directly or indirectly, to stimulate the inductive orientation or movement of such cell populations. Preferably, the protein or peptide has the ability to directly stimulate induced cell migration. Whether a particular protein has chemotactic activity for a cell population can be readily determined using any of the known assays for cell chemotaxis of such proteins or peptides. The activity of the protein of the present invention can be measured, among other means, by the following method: Assay for chemotactic activity (identifying proteins that induce or interfere with chemotaxis) The ability to induce the migration of cells through the
It consists of an assay that measures the ability to induce the adhesion of one cell population to another. Suitable migration and adhesion assays include, but are not limited to, those described below: Current Protocols in Immunology, Ed by JE Colig
an, AM Kruisbeek, DH Margulies, EM Shevach, W. Strober, Pub. Green
ePublishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of
alpha and beta Chemokincs 6.12.1-6.12.28; Taub et al., J. Clin. Invest. 95: 1.
370-1376, 1995; Lind et al., APMIS 103: 140-146, 1995; Mueller et al. Eur. J. Immun.
ol. 25: 1744-1748; Gruber et al., J. of Immunol. 152: 5860-5867, 1994; Johnsto.
n et al., J. of Immunol, 153: 1762-1768, 1994. <Assay for Control of Blood Coagulation by GENSET Protein or its Fragment> The protein encoded by the cDNA of the present invention or its fragment can also be evaluated for its effect on blood coagulation. Many assays for such activity, including those published in the following references, are known to those of skill in the art and are incorporated herein by reference: Linet et al., J. Clin. Pharmacol. 26: 131-140, 1986; Burdick et al., Thrombosis Res. 45:41.
3-419, 1987; Humphrey et al., Fibrinolysis 5: 71-79 (1991); Schaub, Prostagla.
ndins 35: 467-474, 1988. The proteins involved in the control of blood coagulation can then be dispensed as pharmaceuticals and used to treat clinical conditions in which control of blood coagulation would be beneficial. For example, the proteins of the invention may exhibit hemostatic or thrombolytic activity. As a result, such proteins are associated with a variety of coagulation disorders (including inherited disorders such as hemophilia).
Are expected to be useful in the treatment of, and potentiate other hemostatic events in the treatment of wounds due to coagulation and trauma, surgery or other causes. In addition, the proteins of the invention may be useful in inhibiting the dissolution or formation of thrombosis, and in the treatment and prevention of medical conditions resulting therefrom, such as cardiac and central nervous system vascular infarction (eg stroke). Genes encoding these proteins, or nucleic acids that control the expression of these proteins can also be introduced into a suitable host cell to increase or decrease expression of the proteins as desired, as further detailed in.

Assay for Involvement of GENSET Proteins or Fragments Thereof in Receptor / Ligand Interactions The proteins encoded by the cDNA of the invention or fragments thereof are evaluated for their involvement in receptor / ligand interactions. You can also Many such assays for activity include the assays of the references described below,
Known to those of ordinary skill in the art and incorporated herein by reference: Curren
Chapter 7.28 of the Protocols in Immunology (Measurement of Cellular Adhesion un
der Static Conditions 7.28.1-7.28.22), JE Coligan et al., Eds. Greene Pub
lishing Associates and Wiley-Interscience; Takai et al., Proc. Natl. Acad. Sc
i. USA 84: 6864-6868, 1987; Bierer et al., J. Exp. Med. 168: 1145-1156, 1988; R.
osenstein et al., J. Exp. Med. 169: 149-160, 1989; Stoltenborg et al., J. Immunol.
Methods 175: 59-68, 1994; Stitt et al., Cell 80: 661-670, 1995; Gyuris et al., Cell
75: 791-803, 1993. For example, the proteins of the present invention may exhibit activity as inhibitors, agonists of receptors, receptor ligands or receptor / ligand interactions. Examples of such receptors and ligands include, but are not limited to: cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, cell-
Receptors involved in cell interactions and their ligands (such as cell adhesion molecules (selectins, integrins and their ligands)) and receptor / ligand pairs involved in antigen presentation, antigen recognition and the generation of cellular and humoral immune responses Including but not limited to). In addition, receptors and ligands may be useful in screening potential peptide or small molecule inhibitors of related receptor / ligand interactions. The proteins of the invention (including but not limited to fragments of receptors and ligands) may themselves be useful as inhibitors of receptor / ligand interactions. <Assay for GENSET protein or fragment thereof anti-inflammatory activity> The protein encoded by the cDNA of the present invention or a fragment thereof can also be evaluated for their anti-inflammatory activity. Anti-inflammatory activity stimulates cells involved in the inflammatory response, suppresses or promotes cell-cell interactions (such as cell adhesion), suppresses or promotes chemotaxis of cells involved in the inflammatory process ,
It can be achieved by suppressing or promoting extracellular migration of cells, or by stimulating or suppressing the production of factors that more directly suppress or promote the inflammatory response.
Proteins exhibiting such activity are associated with infection-associated inflammation (septic shock,
Sepsis or systemic inflammatory response syndrome (SIRS), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, clones It can be used for the treatment of inflammatory medical conditions including, but not limited to, those caused by disease or overproduction of cytokines such as TNF or IL-1, including chronic or acute medical conditions. Furthermore, the proteins of the invention may also be useful in the treatment of anaphylaxis and hypersensitivity to antigenic substances or materials.

<Assay for tumor suppressor activity of GENSET protein or fragment thereof> The protein encoded by the cDNA of the present invention or a fragment thereof can also be evaluated for their tumor suppressor activity. In addition to the above-mentioned activities of immunological treatment or prevention of tumors, the proteins of the invention may exhibit other anti-neoplastic activities. The protein can directly or indirectly inhibit tumor growth (eg, via ADCC). A protein is a factor, agent or cell that acts on tumor tissue or tumor precursor tissue, inhibits the formation of tissue necessary to support tumor growth (eg, inhibits angiogenesis), inhibits tumor growth It exhibits its tumor suppressor activity by suppressing, eliminating or suppressing factors, agents or cell types that cause the production of the type or promote tumor growth. The proteins of the invention may additionally exhibit one or more of the following activities or effects: inhibition of growth, infection or function of pathogens, including but not limited to bacteria, viruses, fungi and other parasites, Or killing; height, weight, hair color, eye color,
Includes, but is not limited to, skin, body fat / muscle ratio, or other tissue pigmentation, or the size or shape of organs or body parts (such as enlargement or contraction of breasts, changes in bone shape or shape) Not affected physical features (inhibition or enhancement); effects on biological rhythm or circadian cycle or rhythm; effects on fertility of male or female subjects; dietary fats, lipids, proteins, carbohydrates, vitamins, minerals Effects on metabolism, catabolism, assimilation, processing, utilization, storage or elimination of cofactors or other trophic factors or elements; restriction, appetite, libido, stress, cognition (including cognitive impairment), depression (depressive disorder Effects on behavioral characteristics, including but not limited to) and violent behaviors; providing analgesic or other pain relief effects;
Promotion of differentiation and growth of embryonic stem cells in cell lineages other than hematopoietic cell lineages; hormone or endocrine activity; in the case of enzymes, correction of enzyme deficiency and treatment of deficiency-related diseases; hyperproliferative disorders (eg psoriasis etc.) ); Immunoglobulin-like activity (eg, ability to bind antigen or complement); and enhance immune response against such protein or material or entity cross-reactive with such protein The ability of the vaccine composition to act as an antigen. (Reference) Abbondanzo et al., (1993), Meth. Enzymol., Academic Press, New York, pp 803.
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[0501]

[Table 1]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511]

[Table 2]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[Table 3]

[0518]

[Table 4]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[Table 5a]

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531]

[Table 5b]

[0532]

[0533]

[0534]

[0535]

[0536]

[0537]

[Table 6]

[0538]

[0539]

[0540]

[0541]

[0542]

[0543]

[0544]

[Table 7]

[0545]

[0546]

[0547]

[0548]

[0549]

[0550]

[Table 8]

[0551]

[0552]

[Table 9]

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[Table 10]

[0559]

[0560]

[0561]

[0562]

[0563]

[Table 11]

[0564]

[0565]

[Table 12]

[0566]

[0567]

[0568]

[0569]

[0570]

[Sequence list]

[Brief description of drawings]

[Figure 1]   It is a map of the expression vector pPT.

[Fig. 2]   It is a block diagram which shows an example of a computer system.

FIG. 3 is a flow chart illustrating one embodiment of a process 200 that compares a novel nucleotide or protein sequence to a database sequence to determine the identity between the new sequence and the sequence in the database.

FIG. 4. Process 250 for determining whether two sequences are homologous in a computer
4 is a flowchart illustrating an embodiment of the present invention.

FIG. 5 is a flow chart illustrating one embodiment of a process 300 for detecting the presence of features in an array.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) C12N 1/19 C12N 1/21 1/21 C12P 21/02 C 5/10 C12Q 1/68 A C12P 21 / 02 C12N 15/00 ZNAA C12Q 1/68 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM , KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD , GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG , US, UZ, VN, YU, ZA, ZW (72) Inventor Jobert, Severin France, F-75010, Ampasset Renault, 7F Term (reference) 4B024 AA01 AA11 BA31 BA41 BA80 CA04 CA11 DA02 EA04 FA18 GA03 GA11 HA03 HA12 4B063 QA01 QQ08 QQ53 QR32 QR56 QR62 QS25 QS34 4B064 AG01 AG27 CA01 CA19 CA20 CC24 DA01 DA13 4B065 AA93Y AB01 AB05 AC14 BA02 BA08 CA24 CA25 CA44 CA46 4H045 AA10 AA11 AA20 BA10 CA40 DA75 DA76 EA20 EA50 FA72 FA74

Claims (29)

[Claims] 1. An isolated polynucleotide, wherein said polynucleotide is i) any one of the sequences set forth as SEQ ID NOS: 242-482, or a polypeptide encoded by a clonal insert of the deposited clone pool. A polypeptide comprising an amino acid sequence having at least about 80% identity to any one of the sequences,
Or ii) a polynucleotide comprising a nucleic acid sequence encoding a biologically active fragment of said polypeptide. 2. The method of claim 1, wherein the polypeptide comprises any one of the sequences set forth as SEQ ID NOS: 242-482 or one of the polypeptide sequences encoded by the clone inserts of the deposited clone pool. The described polynucleotide. 3. The polynucleotide of claim 1, wherein the polypeptide comprises a signal peptide. 4. The polynucleotide of claim 1, wherein the polypeptide is a mature protein. 5. The nucleic acid sequence according to any one of the sequences set forth in SEQ ID NOs: 1-241 or any one of the sequences of the clone inserts of the deposited clone pool, for at least about 100 contiguous nucleotides thereof. The polynucleotide of claim 1, having at least about 80% identity. 6. The polynucleotide according to any one of the sequences shown as SEQ ID NOs: 1-241,
Alternatively, it hybridizes under stringent conditions with a polynucleotide containing any one of the sequences of the clone inserts of the deposited clone pool.
The polynucleotide according to. 7. The nucleic acid sequence comprises any one of the sequences set forth in SEQ ID NOS: 1-241 or any one of the clone insert sequences of the deposited clone pool.
The polynucleotide according to. 8. The polynucleotide of claim 1, wherein the polypeptide is operably linked to a promoter. 9.   An expression vector comprising the polynucleotide of claim 8. 10.   A host cell that is recombinant for the polynucleotide of claim 1. 11.   A non-human transgenic animal comprising the host cell according to claim 10. 12. A method of producing a GENSET polypeptide, said method comprising a) providing a host cell population comprising the polynucleotide of claim 8 and b) in said host cell Culturing said population of host cells under conditions that induce the production of peptides. 13. The method of claim 12, wherein said method further comprises purifying said polypeptide from said population of host cells. 14. A method of producing a GENSET polypeptide, said method comprising: a) supplying a population of cells comprising the polynucleotide of claim 8; b) inducing production of said polypeptide in said cell. Culturing the population of cells under the conditions described above, and c) purifying the polypeptide from the population of cells. 15. An isolated polynucleotide, wherein said polynucleotide is SEQ ID NO: 1.
A nucleic acid sequence having at least about 80% identity to at least about 100 contiguous nucleotides thereof with any one of the sequences shown as ~ 241 or any one of the clone insert sequences of the deposited clone pool. A polynucleotide comprising. 16. A polynucleotide comprising a polynucleotide containing any one of the sequences shown as SEQ ID NOS: 1 to 241 or a sequence of a clone insert of a deposited clone pool under stringent conditions. 16. The polynucleotide of claim 15 which hybridizes. 17. The polynucleotide according to any one of the sequences set forth in SEQ ID NOs: 1-241,
Or containing any one of the clone insert sequences of the deposited clone pool,
The polynucleotide according to claim 15. 18. A bioactive polypeptide encoded by the polynucleotide of claim 15. 19. An isolated polypeptide or biologically active fragment thereof, wherein said polypeptide is any one of the sequences set forth in SEQ ID NOs: 242-482, or a polypeptide encoded by a clonal insert of the deposited clone pool. A polypeptide comprising an amino acid sequence having at least about 80% sequence identity with any one of the sequences. 20. The polypeptide is selectively recognized by an antibody produced against an antigenic polypeptide, or an antigenic fragment thereof, and the antigenic polypeptide has a sequence shown by SEQ ID NOs: 242-482. 20. The polypeptide of claim 19, comprising any one or any one of the polypeptide sequences encoded by the clone inserts of the deposited clone pool. 21. The method of claim 19, wherein the polypeptide comprises any one of the sequences set forth in SEQ ID NOs: 242-482 or one of the polypeptide sequences encoded by the clone inserts of the deposited clone pool. The described polypeptide. 22. The polypeptide of claim 19, wherein the polypeptide comprises a signal peptide. 23.   20. The polypeptide of claim 19, wherein the polypeptide is a mature protein. 24.   An antibody that specifically binds to the polypeptide of claim 19. 25. A method for determining that the GENSET gene is expressed in a mammal, the method comprising: a) supplying a biological sample from the mammal; b) supplying the biological sample; i) strin. Contacting with either a polynucleotide that hybridizes with the polynucleotide according to claim 1 under gent conditions, or ii) a polypeptide that specifically binds to the polypeptide according to claim 19, and c) Detecting whether there is hybridization between the polynucleotide and RNA species in the sample, or whether there is binding of the polypeptide to a protein in the sample. , The detection of said hybridization or said binding indicates that said GENSET gene is expressed in said mammal. 26. The method of claim 25, wherein the polynucleotide is a primer and the hybridization is detected by detecting the presence of an amplification product containing the primer sequence. 27.   26. The method of claim 25, wherein the polypeptide is an antibody. 28. A method of determining that a GENSET gene expression level in a mammal is increased or decreased, the method comprising: a) providing a biological sample from the mammal; and b) in the biological sample. Comparing the amount of the polypeptide of claim 19 or the amount of RNA species encoding the polypeptide to a level detected or predicted from a control sample, An increased amount of the polypeptide or the RNA species in the biological sample compared to the detected or predicted level indicates that the GENSET gene expression level in the mammal is elevated and is detected from the control sample. Or the reduced amount of said polypeptide or said RNA species in said biological sample compared to said predicted level results in a reduced level of said GENSET gene expression in said mammal. How to indicate that you are doing. 29. A method of identifying a candidate modulator of a GENSET polypeptide, the method comprising: a) contacting the polypeptide of claim 18 with a test compound; and b) adding the compound to the polypeptide. Determining that it specifically binds, and detecting that the compound specifically binds to the polypeptide indicates that the compound is a candidate modulator of the GENSET polypeptide.